Holmes was born Herman Webster Mudgett in Gilmanton, New Hampshire, on May 16, 1861, to Levi Horton Mudgett and Theodate Page Price, both of whom were descended from the first English immigrants in the area. Mudgett was his parents' third-born child; he had an older sister Ellen, an older brother Arthur, a younger brother Henry and a younger sister Mary. Holmes's father was from a farming family, and at times he worked as a farmer, trader and house painter; his parents were devout Methodists. Later attempts to fit Holmes into the patterns seen in modern serial killers have described him torturing animals and suffering from abuse at the hands of a violent father, but contemporary and eyewitness accounts of his childhood do not provide proof of either.

At the age of 16, Holmes graduated from high school and took teaching jobs in Gilmanton and later in nearby Alton. On July 4, 1878, he married Clara Lovering in Alton; their son, Robert Lovering Mudgett, was born on February 3, 1880, in Loudon, New Hampshire. As an adult, Robert became a certified public accountant, and served as city manager of Orlando, Florida.

Holmes enrolled in the University of Vermont in Burlington at age 18, but was dissatisfied with the school and left after only one year. In 1882, he entered the University of Michigan's Department of Medicine and Surgery and graduated in June 1884 after passing his examinations. While enrolled, he worked in the anatomy lab under Professor Herdman, then the chief anatomy instructor. Holmes had previously apprenticed in New Hampshire under Dr. Nahum Wight, a noted advocate of human dissection. Years later, when Holmes was suspected of murder and claimed to be nothing but an insurance fraudster, he admitted to using cadavers to defraud life insurance companies several times in college.

Holmes arrived in Chicago in August 1886 and came across Elizabeth S. Holton's drugstore at the southwest corner of South Wallace Avenue and West 63rd Street in Englewood. Holton gave Holmes a job, and he proved himself to be a hardworking employee, eventually buying the store. Although several books portray Holton's husband as an old man who quickly vanished along with his wife, Dr. Holton was actually a fellow Michigan alumnus, only a few years older than Holmes, and both Holtons remained in Englewood throughout Holmes' life and survived well into the 20th century; the idea that Holmes killed them is strictly fiction.

Housemates described Holmes as treating Clara violently, and in 1884, before his graduation, she moved back to New Hampshire and later wrote that she knew little of him afterwards. After he moved to Mooers Forks, New York, a rumor spread that Holmes had been seen with a little boy who later disappeared. Holmes claimed the boy went back to his home in Massachusetts. No investigation took place and Holmes quickly left town. He later traveled to Philadelphia and eventually got a job as a keeper at Norristown State Hospital, but quit after a few days. Subsequently, he took a position at a drugstore in Philadelphia, but while he was working there, a boy died after taking Medicine that was purchased at the store. Holmes denied any involvement in the child's death and immediately left the city. Right before moving to Chicago, he changed his name to Henry Howard Holmes to avoid the possibility of being exposed by victims of his previous scams. In his confession following his arrest, Holmes claimed that he had killed his former medical school classmate, Dr. Robert Leacock, in 1886 for insurance money; Dr. Leacock, however, died in Watford, Ontario in Canada on October 5, 1889. In late 1886, while still married to Clara, Holmes married Myrta Belknap (b. October 1862 in Pennsylvania) in Minneapolis, Minnesota. He filed for divorce from Clara a few weeks after marrying Myrta, alleging infidelity on her part, but the claims could not be proven and the suit went nowhere. Surviving paperwork indicated that she probably was never even informed of the suit. In any case the divorce was never finalized; it was dismissed June 4, 1891, on the grounds of "want of prosecution". Holmes had a daughter with Myrta, Lucy Theodate Holmes, who was born on July 4, 1889, in Englewood, Chicago; as an adult, Lucy became a public schoolteacher. Holmes lived with Myrta and Lucy in Wilmette, Illinois, and spent most of his time in Chicago tending to Business. Holmes married Georgiana Yoke on January 17, 1894, in Denver, Colorado, while still married to both Clara and Myrta.

One of Holmes' early murder victims was his mistress, Julia Smythe. She was the wife of Ned (Icilius) Conner, who had moved into Holmes' building and began working at his pharmacy's jewelry counter. After Conner found out about Smythe's affair with Holmes, he quit his job and moved away, leaving Smythe and her daughter Pearl behind. Smythe gained custody of Pearl and remained at the hotel, continuing her relationship with Holmes. Julia and Pearl disappeared on Christmas Eve of 1891, and Holmes later claimed that she had died during an abortion, though what truly happened to the two was never confirmed. Another likely Holmes paramour, Emeline Cigrande, began working in the building in May 1892, and disappeared that December. Another woman who vanished, Edna Van Tassel, is also believed to have been one of Holmes' victims.

Devil in the White City, an upcoming film starring Leonardo DiCaprio as Holmes, is set to be directed by Martin Scorsese and written by Billy Ray, based on the book of the same name. The film will follow Daniel H. Burnham's construction of the 1893 World's Fair, as well as Holmes' building of his hotel.

Frank Geyer, a Philadelphia police detective assigned to investigate Holmes and find the three missing children, located the decomposed bodies of the two Pitezel girls in the cellar of the Toronto home. Detective Geyer wrote, "The deeper we dug, the more horrible the odor became, and when we reached the depth of three feet, we discovered what appeared to be the bone of the forearm of a human being." Geyer then went to Indianapolis, where Holmes had rented a cottage. Holmes was reported to have visited a local pharmacy to purchase the drugs which he used to kill Howard Pitezel, and a repair shop to sharpen the knives he used to chop up the body before he burned it. The boy's teeth and bits of bone were discovered in the home's chimney. Holmes' murder spree finally ended when he was arrested in Boston on November 17, 1894, after being tracked there from Philadelphia by the Pinkertons. He was held on an outstanding warrant for horse theft in Texas, as the authorities had become more suspicious at this point and Holmes appeared poised to flee the country in the company of his unsuspecting third wife.

In October 1895, Holmes was put on trial for the murder of Benjamin Pitezel, and was found guilty and sentenced to death. By then, it was evident that Holmes had also murdered the Pitezel children. Following his conviction, Holmes confessed to 27 murders in Chicago, Indianapolis, and Toronto (though some persons he "confessed" to murdering were, in fact, still living), and six attempted murders. Holmes was paid $7,500 (worth $221,000 today) by the Hearst newspapers in exchange for his confession, which was quickly found to be mostly nonsense. Holmes gave various contradictory accounts of his life, initially claiming innocence and later that he was possessed by Satan. His propensity for lying has made it difficult for researchers to ascertain the truth on the basis of his statements. While writing his confessions in prison, Holmes mentioned how drastically his facial appearance had changed since his imprisonment. He described his new, grim appearance as "gruesome and taking a Satanical Cast," and wrote that he was now convinced that after everything that he had done, he was beginning to resemble the Devil.

On May 7, 1896, Holmes was hanged at Moyamensing Prison, also known as the Philadelphia County Prison, for the murder of Pitezel. Until the moment of his death, Holmes remained calm and amiable, showing very few signs of fear, anxiety or depression. Despite this, he asked for his coffin to be contained in cement and buried 10 feet deep, because he was concerned grave Robbers would steal his body and use it for dissection. Holmes' neck did not snap; he instead was strangled to death slowly, twitching for over 15 minutes before being pronounced dead 20 minutes after the trap had been sprung.

On New Year's Eve 1909, Hedgepeth, who had been pardoned for informing on Holmes, was shot and killed by police officer Edward Jaburek during a holdup at a Chicago saloon.

On March 7, 1914, the Chicago Tribune reported that, with the death of Quinlan, the former caretaker of the Castle, "the mysteries of Holmes' Castle" would remain unexplained. Quinlan had committed suicide by taking strychnine. His body was found in his bedroom with a note that read, "I couldn't sleep." Quinlan's surviving relatives claimed that he had been "haunted" for several months and was suffering from hallucinations. The Castle itself was mysteriously gutted by fire in August 1895. According to a newspaper clipping from The New York Times, two men were seen entering the back of the building between 8 and 9 p.m. About a half an hour later, they were seen exiting the building, and rapidly running away. Following several explosions, the Castle went up in flames. Afterwards, investigators found a half-empty gas can underneath the back steps of the building. The building survived the fire and remained in use until it was torn down in 1938. The site is currently occupied by the Englewood branch of the United States Postal Service.

While he confessed to 27 murders, only nine could be plausibly confirmed and several of the people whom he claimed to have murdered were still alive. He is commonly said to have killed as many as 200, though this figure is only traceable to 1940s pulp magazines. Many victims were said to have been killed in a mixed-use building which he owned, located about 3 miles (4.8 km) west of the 1893 World's Fair: Columbian Exposition, supposedly called the World's Fair Hotel (informally called "The Murder Hotel"), though evidence suggests that the hotel portion was never truly open for Business.

The case was notorious in its time and received wide publicity in the international press. Interest in Holmes's crimes was revived in 2003 by Erik Larson's The Devil in the White City: Murder, Magic, and Madness at the Fair That Changed America, a best-selling nonfiction book that juxtaposed an account of the planning and staging of the World's Fair with Holmes's story. His story had been previously chronicled in The Torture Doctor by David Franke (1975), The Scarlet Mansion by Allan W. Eckert (1985) and Depraved: The Shocking True Story of America's First Serial Killer by Harold Schechter (1994), as well as "The Monster of Sixty-Third Street" chapter in Gem of the Prairie: An Informal History of the Chicago Underworld by Herbert Asbury (1940, republished 1986). Asbury's account drew heavily on 1890s tabloids and included several claims – such as the "200 victims" figure, Holmes killing Dr. Holton and torture equipment found in the castle – that, according to Selzer, were the products of his own imagination. However, Asbury's account was a major foundation for later retellings of Holmes, including Larson's, which quoted several portions of Asbury's account verbatim. The 1974 novel American Gothic by horror Writer Robert Bloch was a fictionalized version of the story of H. H. Holmes. In 2003, cartoonist/illustrator Rick Geary published a graphic novel about Holmes titled The Beast of Chicago: The Murderous Career of H. H. Holmes. Selzer's comprehensive 2017 biography, H. H. Holmes: The True History of the White City Devil, attempted to separate fact from fiction, and to trace how the story grew.

A documentary film on Holmes, H. H. Holmes: America's First Serial Killer, was released in 2004, narrated by Tony Jay. The Producer and Director of the film, John Borowski, also wrote a book on Holmes titled The Strange Case of Dr. H. H. Holmes.

Episode 3 of the first season of the History Channel's Haunted History TV series, airing in July 2013, was dedicated to Holmes and the Murder Castle.

A three-part miniseries, titled The Murder Castle, premiered October 28, 2017 on Investigation Discovery.