Dvořák was born in Nelahozeves near Prague, in the Austrian Empire, and was the eldest son of František Dvořák (1814–94) and his wife, Anna, née Zdeňková (1820–82). František worked as an innkeeper, a professional player of the zither, and a butcher. Anna was the daughter of Josef Zdeněk, the bailiff of the Prince of Lobkowicz. Anna and František married on 17 November 1840. Dvořák was the first of fourteen children, eight of whom survived infancy. Dvořák was baptized as a Roman Catholic in the village's church of St. Andrew. Dvořák's years in Nelahozeves nurtured his strong Christian faith and the love for his Bohemian heritage that so strongly influenced his music. In 1847, Dvořák entered primary school and was taught to play violin by his Teacher Joseph Spitz. He showed early talent and skill, playing in a village band and in church. František was pleased with his son's gifts. At the age of 13, through the influence of his father, Dvořák was sent to Zlonice to live with his uncle Antonín Zdenĕk in order to learn the German language. His first composition, the Forget-Me-Not Polka in C (Polka pomněnka) was written possibly as early as 1855.

Dvořák had been an admirer of Wagner's music since 1857. Late in life, he said that Wagner "was so great a genius that he was capable of doing things that were beyond the reach of other composers". Wagner especially influenced Dvořák's operas, but also some orchestral pieces. According to Clapham, the theme of the Andante Sostenuto from his fourth symphony "could almost have come directly out of Tannhäuser".

In 1858, he joined Karel Komzák's orchestra, with whom he performed in Prague's restaurants and at balls. The high professional level of the ensemble attracted the attention of Jan Nepomuk Maýr, who engaged the whole orchestra in the Bohemian Provisional Theater Orchestra. Dvořák played viola in the orchestra beginning in 1862. Dvořák could hardly afford concert tickets, and playing in the orchestra gave him a chance to hear music, mainly operas. In July 1863, Dvořák played in a program devoted to the German Composer Richard Wagner, who conducted the orchestra. Dvořák had had "unbounded admiration" for Wagner since 1857. In 1862, Dvořák had begun composing his first string quartet. In 1864, Dvořák agreed to share the rent of a flat located in Prague's Žižkov district with five other people, who also included Violinist Mořic Anger and Karel Čech, who later became a singer. In 1866, Maýr was replaced as chief Conductor by Bedřich Smetana. Dvořák was making about $7.50 a month. The constant need to supplement his income pushed him to give piano lessons. It was through these piano lessons that he met his Future wife. He originally fell in love with his pupil and colleague from the Provisional Theater, Josefína Čermáková, for whom he apparently composed the song-cycle "Cypress Trees". However, she never returned his love and ended up marrying another man. In 1873 Dvořák married Josefina's younger sister, Anna Čermáková (1854–1931). They had nine children together, three of whom died in infancy.

Over a period of almost 30 years, Dvořák's output of chamber music was prolific and diverse, including more than 40 works for ensembles with strings.
In 1860 just after he finished his education at the Organ school, Dvořák composed his String Quintet No. 1 in A minor, Op. 1. Two more would follow, of which the String Quintet No. 2 in G major, Op. 77 from early 1875, is noteworthy for the use of a double bass. It was written for a chamber music competition sponsored by the Umělecká beseda (Artistic Circle), where it was unanimously awarded the prize of five ducats for the "distinction of theme, the technical skill in polyphonic composition, the mastery of form and the knowledge of the instruments" displayed. The String Quintet No.3 in E♭ major, Op. 97, with a second viola added, was written near the end of his output for chamber ensemble during his American period in 1893, when he spent a summer holiday in Spillville, Iowa.

Dvořák called his String Quintet in A Minor (1861) his Opus 1, and his First String Quartet (1862) his Opus 2, although the chronological Burghauser Catalogue numbers these as B.6 and B.7, showing five earlier compositions without opus numbers. In the early 1860s, Dvořák also made his first symphonic attempts, some of which he self-critically burned. The manuscript of a symphony in C minor without opus number, B.9, composed in 1865, was preserved. This symphony has come to be numbered as Dvořák's First (see under "Works"). His first composing attempts passed without critical reception or public performances. His compositions up through 1870, according to the Burghauser Catalogue either had no known premieres, or were premiered in 1888 or later. For Example, the Third String Quartet, B.18, was written in about 1869 but first published in 1964 and premiered in 1969. In 1870, he composed his first opera, Alfred, over the course of five months from May to October. Its overture was first publicly performed as late as 1905, and the full opera only in 1938.

His most popular quartet is his twelfth, the American, Op. 96. He also composed two piano quintets, both in A major, of which the second, Op. 81, is the better known. He left a Terzetto for two violins and viola (Op. 74); two piano quartets, a string sextet; Op. 48; and four piano trios, including the Piano Trio No. 4 (subtitled Dumky), Op. 90. He also wrote a set of Bagatelles, Op. 47, for the unusual instrumentation of two violins, cello, and harmonium, two waltzes for string quartet, and a set of twelve love songs arranged for quartet, taken from his set of 18 songs originally composed in 1865 entitled Cypresses.

While a large number of Dvořák's works were given opus numbers, these did not always bear a logical relationship to the order in which they were either written or published. To achieve better sales, some publishers such as N. Simrock preferred to present budding composers as being well established, by giving some relatively early works much higher opus numbers than their chronological order would merit. In other cases, Dvořák deliberately provided new works with lower opus numbers to be able to sell them outside contract obligations to other publishers. An Example is the Czech Suite which Dvořák didn't want to sell to Simrock, and had published with Schlesinger as Op. 39 instead of Op. 52. In this way it could come about that the same opus number was given to more than one of Dvořák's works; for Example the opus number 12, which was assigned, successively, to: the opera King and Charcoal Burner (1871), the Concert Overture in F (1871, derived from the opera), the String Quartet No. 6 in A minor (1873), the Furiant in G minor for piano (1879), and the Dumka in C minor for piano (1884). In yet other cases, a work was given as many as three different opus numbers by different publishers.

In November 1872, Dvořák's Piano Quintet in A major, Op. 5, was performed in Prague, by a "splendid team of players" organized by Procházka. It was his first piece played in a concert. In March 1873, his Czech patriotic cantata The Heirs of the White Mountain was performed by the Prague Hlahol Choral Society of 300 Singers (conducted by his friend and supporter Karel Bendl) to a warm response from both audience and critics, making it an "unqualified success". Dvořák's compositions were first coming to be recognized in Prague.

Symphony No. 3 in E-flat major, Op. 10 (c. 1873), shows the impact of Dvořák's recent acquaintance with the music of Richard Wagner. This influence is less evident in Symphony No. 4 in D minor, Op. 13, except for the start of the second movement.

Clapham gives the official report for the 1874 prize, saying Dvořák was a relatively impoverished music Teacher who "has submitted 15 compositions, among them symphonies, which display an undoubted talent...The applicant... deserves a grant to ease his straitened circumstances and free him from anxiety in his creative work." It says he had not yet owned a piano. Before being married, he had lodged with five other men, one of whom owned a small "spinet" piano.

In 1875, the year his first son was born, Dvořák composed his second string quintet, his 5th Symphony, Piano Trio No. 1, and Serenade for Strings in E. He again entered but this time did not win the Austrian State Prize. He did win it in 1876, and finally felt free to resign his position as an organist. In 1877 he wrote the "Symphonic Variations" and Ludevít Procházka conducted its premiere in Prague.

A work that does not fit into any of the above categories is the Symphonic Variations of 1877. Orchestral variations on an original theme, composed as a freestanding work, were a rather unusual genre. Originally unsuccessful and revived only after ten years, it has since established itself in the repertoire.

From other important works, that show also the influence of Czech folk music, both in terms of rhythms and of melodic shapes, perhaps the best known examples are the two sets of Slavonic Dances, written in two series. The first book, Op. 46 (1878), is predominantly Czech in respect to the forms represented. They were created for piano duet (one piano, four hands), but Dvořák proceeded to orchestrate the entire set, completing that version the same year. The second book, Op. 72 (also composed originally for piano four hands), which came along eight years later, includes forms native to such other Slavic lands as Serbia, Poland and Ukraine, although some "merge characteristics of more than one dance". Dvořák did not use actual folk tunes in his dances, but created his own themes in the authentic style of traditional folk music, using only rhythms of original folk dances.

Many of Dvořák's compositions, such as the Slavonic Dances and his large collection of songs, were directly inspired by Czech, Moravian, and other Slavic traditional music. As the basis for his works, Dvořák frequently used Slavic folk dance forms including the skočná; the Bohemian odzemek, furiant, sousedská, and špacirka; the Polish mazurka and polonaise; the Yugoslav Kolo; and folk song forms of Slavic peoples, including the Ukrainian dumka. His 16 Slavonic Dances, Op. 46, which first brought him a wide reputation, and Op. 72, include at least one of each of these forms. He also wrote an orchestral Polonaise (1879). He named the third movement of his 6th Symphony as "Scherzo (Furiant)". His Dumky Trio is one of his best-known chamber works, and is named for the Dumka, a traditional Slavic and Polish genre. His major works reflect his heritage and love for his native land. Dvořák followed in the footsteps of Bedřich Smetana, the creator of the modern Czech musical style.

Within a year after completing his first string quintet, Dvořák completed his String Quartet No. 1 in A major, Op. 2, the first of his fourteen string quartets. For some time Dvořák was very tentative in his approach to quartets. In the 1880s Dvořák made a list of compositions he had destroyed, which lists two quartets and 2 other quartets. He may well have destroyed the scores, but only after the individual instrumental parts had been copied out. The number of errors in the parts makes it highly unlikely that he actually had them played. The quartets numbered 2 to 4 were probably composed between 1868 and 1870 and show the strong influence of the music of Richard Wagner. Dvořák kept the manuscripts of these quartets but did not give them opus numbers. They have numbers B.17, B.18, and B.19 in the Burghauser catalog. An Andante religioso from his fourth quartet was used five years later in his second string quintet Op. 77, as a second movement named Intermezzo: Nocturne, making this initially a five-movement composition, although he later withdrew this second movement, and later still reworked it variously, resulting in the Nocturne for Strings in B major, Op. 40 (B. 47). The two Quartets he wrote in 1873 (number 5, B37 and number 6, B40) show a stronger sense of form.

Hans Richter asked Dvořák to compose his Symphony No. 6 for the Vienna Philharmonic, intending to premiere it in December 1880. However, Dvořák later discovered that, despite this intention, members of the orchestra objected to performing works by the Composer in two consecutive seasons, due to "anti-Czech feeling". Adolf Čech therefore conducted the premiere of the symphony at a concert of the Philharmonia society (in Czech: spolek Filharmonie, predecessor of the Czech Philharmonic) on 25 March 1881, in Prague. Richter did eventually conduct the piece in London in 1882 and always retained an interest in Dvořák's compositions.

Dvořák's Stabat Mater (1880) was performed and very well received at the Royal Albert Hall in London on 10 March 1883, conducted by Joseph Barnby. The success "sparked off a whole series of performances in England and the United States", a year ahead of appreciation in Germany and Austria. Dvořák was invited to visit Britain where he appeared to great acclaim in 1884. The London Philharmonic Society commissioned Dvořák to conduct concerts in London, and his performances were well received there. In response to the commission, Dvořák wrote his Symphony No. 7 and conducted its premiere at St. James's Hall on 22 April 1885. On a visit later in 1885, Dvořák presented his cantata The Spectre's Bride, in a concert on 27 August. He had arrived a week early to conduct rehearsals of the chorus of 500 voices and orchestra of 150. The performance was "a greater triumph than any" Dvořák "had had in his life up to that time...following this phenomenal success, choral societies in the English-speaking countries hastened to prepare and present the new work." Dvořák visited Britain at least eight times in total, conducting his own works there. In 1887, Richter conducted the Symphonic Variations in London and Vienna to great acclaim (they had been written ten years earlier and Dvořák had allowed them to languish after initial lack of interest from his publishers). Richter wrote to Dvořák of the London performance, "at the hundreds of concerts I have conducted during my life, no new work has been as successful as yours."

The cantata The Spectre's Bride, Op. 69, B. 135, performed in 1885 at the Birmingham, England, Musical Festival, was the greatest success in Dvořák's career up to that point.

Despite Dvořák's newfound success, a February 1888 performance of Stabat Mater in Vienna fell victim to more anti-Czech feeling and what the Composer called "destructive criticism". He heartily thanked Richter for his "courage and devoted sympathy". In 1890, influenced by Pyotr Ilyich Tchaikovsky, Dvořák also visited Russia, and conducted performances of his music in Moscow and St. Petersburg. In 1891, Dvořák received an honorary degree from the University of Cambridge, and was offered a position at the Prague Conservatory as professor of composition and instrumentation. At first he refused the offer, but then later accepted; this change of mind was seemingly a result of a quarrel with his publisher Simrock over payment for his Eighth Symphony. Dvořák's Requiem was premiered later that year in Birmingham at the Triennial Music Festival.

Antonín Dvořák composed his Requiem in 1890, at the beginning of the peak period of his career. Dvořák was a deeply religious man, and this work reflects his faith and spirituality. The premiere of the work took place on 9 October 1891 in Birmingham, conducted by Dvořák himself, and was "very successful". It had an outstanding success in Boston 30 November 1892: "the Composer was frequently applauded between numbers and given a most enthusiastic ovation at the end.". In Vienna it was greeted, belatedly, in 1901: "The Vienna performance in March 1901 was a triumph of Dvořák's music, as if the Viennese public wished thereby to make up for their earlier, sometimes cool reception of his works."

The Mass in D major (originally numbered as Op. 76, finally as Op. 86) was originally intended for organ, solo voices and small choir. The work was given its final shape in the year 1892 when, in response to a request from the Novello publishers of London, Dvořák arranged his Mass for a symphony orchestra.

Symphony No. 9 in E minor, Op. 95, is also known by its subtitle, From the New World, or as the New World Symphony. Dvořák wrote it between January and May 1893, while he was in New York. At the time of its first performance, he claimed that he used elements from American music such as spirituals and Native American music in this work, but he later denied this. Reacting to American racism, he wrote in an article published in the New York Herald on 15 December 1893, "[In the 9th symphony] I have simply written original themes embodying the peculiarities of the Indian music." Neil Armstrong took a recording of the New World Symphony to the Moon during the Apollo 11 mission, the first Moon landing, in 1969, and in 2009 it was voted the favourite symphony in a poll run by ABC Classic FM in Australia.

The song cycle of 10 Biblical Songs, Op. 99, B. 185, was written in March 1894. Around that time Dvořák was informed of the death of the famous Conductor, and his close personal friend, Hans von Bülow. Just a month earlier, he had been grieved to hear that his father was near death, far away in Bohemia. Dvořák consoled himself in the Psalms. The resulting work, considered the finest of his song cycles, is based on the text of the Czech Bible of Kralice. As fate would have it, his father expired 28 March 1894, two days after the completion of the work.

Dvořák returned from the United States on 27 April 1895 with his wife and Otakar Berger, and took care to avoid spreading the news about his return. However, after a performance of Dimitrij at the National Theater on 19 May, Dvořák fled to the family country cottage in Vysoká. Dvořák's first love and later sister-in-law, Josefina Kaunitzová, née Čermáková, died in May 1895. He and she had maintained friendly relations over the years. After her death he revised the coda of his Cello Concerto in her memory. During Dvořák's final years, he concentrated on composing opera and chamber music. In November 1895, he resumed his professorship at the Prague Conservatory. Between 1895 and 1897, he completed his string quartets in A-flat major and G major, and also worked on the cycle of symphonic poems inspired by the collection Kytice by Karel Jaromír Erben. As seen in Burghauser's 1960 Catalogue, Dvořák wrote his five Symphonic Poems in 1896, but after that completed few works per year, mainly operas: Jakobín in 1896, nothing in 1897, only The Devil and Kate in 1898–99, Rusalka in 1900, two songs and "Recitatives" in 1900/01, and finally the opera Armida in 1902–03. Rusalka became the most popular of all Dvořák's ten operas and gained an international reputation (below under Works, Operas).

The Concerto for Cello and Orchestra in B minor, Op. 104 was the last composed of Dvořák's concerti. He wrote it in 1894–1895 for his friend the Cellist Hanuš Wihan. Wihan and others had asked for a cello concerto for some time, but Dvořák refused, stating that the cello was a fine orchestral instrument but completely insufficient for a solo concerto. Dvořák composed the concerto in New York while serving as the Director of the National Conservatory. In 1894 Victor Herbert, who was also teaching at the Conservatory, had written a cello concerto and presented it in a series of concerts. Dvořák attended at least two performances of Victor Herbert's cello concerto and was inspired to fulfill Wihan's request for a cello concerto. Dvořák's concerto received its premiere in London on 16 March 1896, with the English Cellist Leo Stern. The reception was "enthusiastic". Brahms said of the work: "Had I known that one could write a cello concerto like this, I would have written one long ago!" Agreeing with Schonberg, the Cellist and author Robert Battey wrote "I believe it to be the greatest of all cello concertos...an opinion shared by most cellists". A compiler of discographies of Dvořák's music wrote that his is the "king" of cello concertos.

In 1897 Dvořák's daughter Otilie married his student, the Composer Josef Suk. In the same year, Dvořák visited Brahms on his deathbed and attended his funeral on 6 April 1897. In November Dvořák was appointed a member of the jury for the Viennese Artists’ Stipendium. He was informed in November 1898 that Emperor Franz Joseph I of Austria-Hungary would award him a gold medal for Litteris et Artibus, the ceremony taking place before an audience in June 1899. On 4 April 1900 Dvořák conducted his last concert with the Czech Philharmonic, performing Brahms’ Tragic Overture, Schubert's "Unfinished" Symphony, Beethoven's 8th Symphony. and Dvořák's own symphonic poem The Wild Dove. In April 1901, The Emperor appointed him a member of the Austro-Hungarian House of Lords, along with the leading Czech poet Jaroslav Vrchlický. Dvoŕák also succeeded Antonín Bennewitz as Director of the Prague Conservatory from November 1901 until his death. Dvořák's 60th birthday was celebrated as a national event. First, around the actual date, six of his operas and the oratorio St. Ludmila were performed in Prague, but Dvořák was away in Vienna; then in November 1901 came the "postponed official birthday party... In many towns all over Bohemia and Moravia, the Czech people celebrated his birthday."

The oratorio Saint Ludmila was a huge success in Bohemia and Moravia, sung at events in Dvořák's honor in 1901 and 1904. Its text, in Czech, may have limited its audience among non-Czech speakers. The piece had a considerable success in England in October 1886, with an audience on the 15th "in raptures... the critics praised the music in the warmest terms", and on the 29th, there was a "large and equally enthusiastic audience, and once again the critics were full of praise", but a drawback was that the libretto, specifically its translation into English, was "regarded on all sides as unsatisfactory".

In a 1904 interview, Dvořák claimed that opera was 'the most suitable form for the nation'. If this nationalist sentiment was at the heart of his opera compositions, he also struggled to find a style straddling Czech traditional melody and the grand opera style of Giacomo Meyerbeer, which he experienced as lead viola player in the orchestra of Prague's Provisional Theatre between 1862 and 1871, and whose influence is very evident in his works such as Vanda and Dimitrij. His later interest in the music of Richard Wagner also affected his operas, evident in the very extensive rewrite of Dmitrij in 1894, following its failure at Vienna.

The sequential numbering of his symphonies has also been confused: (a) they were initially numbered by order of publication, not composition; (b) the first four symphonies to be composed were published after the last five; and (c) the last five symphonies were not published in order of composition. This explains why, for Example, the New World Symphony was originally published as No. 5, was later known as No. 8, and definitively renumbered as No. 9 in the critical editions published in the 1950s.

1980 film Concert at the End of Summer is based on Dvořák's life. Dvořák was played by Josef Vinklář. 2012 television film The American Letters focuses on Dvořák's love life. Dvořák is played by Hynek Čermák.

Dvořák's New York home was located at 327 East 17th Street, near the intersection of what is today called Perlman Place. It was in this house that both the B minor Cello Concerto and the New World Symphony were written within a few years. Despite protests, from Czech President Václav Havel amongst others, who wanted the house preserved as a historical site, it was demolished in 1991 to make room for a Beth Israel Medical Center residence for people with AIDS. To honor Dvořák, however, a statue of him was erected in nearby Stuyvesant Square.

Of all his operas, only Rusalka, Op. 114, which contains the well-known aria "Měsíčku na nebi hlubokém" ("Song to the Moon"), is played on contemporary opera stages with any frequency outside the Czech Republic. This is attributable to their uneven invention and libretti, and perhaps also their staging requirements – The Jacobin, Armida, Vanda and Dimitrij need stages large enough to portray invading armies.

During Dvořák's life, only five of his symphonies were widely known. The first published was his sixth, dedicated to Hans Richter. After Dvořák's death, research uncovered four unpublished symphonies, of which the manuscript of the first had even been lost to the Composer himself. This led to an unclear situation in which the New World Symphony has alternately been called the 5th, 8th and 9th. This article uses the modern numbering system, according to the order in which the symphonies were written.

Brahms continued to try to "clear a path for" Dvořák, "the only contemporary whom he considered really worthy". While Dvořák was in America, Simrock was still publishing his music in Germany, and Brahms corrected proofs for him. Dvořák said it was hard to understand why Brahms would "take on the very tedious job of proofreading. I don’t believe there is another musician of his stature in the whole world who would do such a thing."