Mayr was the second son of Helene Pusinelli and Dr. Otto Mayr. His Father was a jurist (District Prosecuting Attorney at Würzburg) but took an interest in natural history and took the children out on field trips. He learnt all the local birds in Würzburg from his elder brother Otto. He also had access to a natural history magazine for amateurs, Kosmos. His Father died just before he was thirteen. The family then moved to Dresden and he studied at the Staatsgymnasium (“Royal Gymnasium” until 1918) in Dresden-Neustadt and completed his high school education there. In April 1922, while still in high school, he joined the newly founded Saxony Ornithologists’ Association. Here he met Rudolf Zimmermann, who became his ornithological mentor. In February 1923, Mayr passed his high school examination (Abitur) and his mother rewarded him with a pair of binoculars.

On 23 March 1923 on the lakes of Moritzburg, the Frauenteich, he spotted what he identified as a red-crested pochard. The species had not been seen in Saxony since 1845 and the local club argued about the identity. Raimund Schelcher (1891–1979) of the club then suggested that Mayr visit his classmate Erwin Stresemann on his way to Greifswald, where Mayr was to begin his medical studies. After a tough interrogation, Stresemann accepted and published the sighting as authentic. Stresemann was very impressed and suggested that, between semesters, Mayr could work as a volunteer in the ornithological section of the museum. Mayr wrote about this event, "It was as if someone had given me the key to heaven." He entered the University of Greifswald in 1923 and, according to Mayr himself, "took the medical curriculum (to satisfy a family tradition) but after only a year, he decided to leave Medicine and enrolled at the Faculty of Biological Sciences." Mayr was endlessly interested in ornithology and "chose Greifswald at the Baltic for my studies for no other reason than that ... it was situated in the ornithologically most interesting area." Although he ostensibly planned to become a physician, he was "first and foremost an ornithologist." During the first semester break Stresemann gave him a test to identify treecreepers and Mayr was able to identify most of the specimens correctly. Stresemann declared that Mayr "was a born systematist". In 1925, Stresemann suggested that he give up his medical studies, in fact he should leave the faculty of Medicine and enrol into the faculty of Biology and then join the Berlin Museum with the prospect of bird-collecting trips to the tropics, on the condition that he completed his doctoral studies in 16 months. Mayr completed his doctorate in ornithology at the University of Berlin under Dr. Carl Zimmer, who was a full professor (Ordentlicher Professor), on 24 June 1926 at the age of 21. On 1 July he accepted the position offered to him at the museum for a monthly salary of 330.54 Reichsmark.

At the International Zoological Congress at Budapest in 1927, Mayr was introduced by Stresemann to banker and naturalist Walter Rothschild, who asked him to undertake an expedition to New Guinea on behalf of himself and the American Museum of Natural History in New York. In New Guinea, Mayr collected several thousand bird skins (he named 26 new bird species during his lifetime) and, in the process also named 38 new orchid species. During his stay in New Guinea, he was invited to accompany the Whitney South Seas Expedition to the Solomon Islands. Also, while in New Guinea, he visited the Lutheran missionaries Otto Thiele and Christian Keyser, in the Finschhafen district; there, while in conversation with his hosts, he uncovered the discrepancies in Hermann Detzner's popular book Four Years Among the Cannibals in German Guinea from 1914 to the Truce, in which Detzner claimed to have seen the interior, discovered several species of flora and fauna, while remaining only steps ahead of the Australian patrols sent to capture him.

He returned to Germany in 1930, and in 1931 he accepted a curatorial position at the American Museum of Natural History, where he played the important role of brokering and acquiring the Walter Rothschild collection of bird skins, which was being sold in order to pay off a blackmailer. During his time at the museum he produced numerous publications on bird taxonomy, and in 1942 his first book Systematics and the Origin of Species, which completed the evolutionary synthesis started by Darwin.

Mayr also greatly influenced the American ornithologist Margaret Morse Nice. Mayr encouraged her to correspond with European ornithologists and helped her in her landmark study on song sparrows. Nice wrote to Joseph Grinnell in 1932, trying to get foreign literature reviewed in the Condor: "Too many American ornithologists have despised the study of the living bird; the magazines and books that deal with the subject abound in careless statements, anthropomorphic interpretations, repetition of ancient errors, and sweeping conclusions from a pitiful array of facts.  ... in Europe the study of the living bird is taken seriously. We could learn a great deal from their writing." Mayr ensured that Nice could publish her two-volume Studies in the Life History of the Song Sparrow. He found her a publisher, and her book was reviewed by Aldo Leopold, Joseph Grinnell, and Jean Delacour. Nice dedicated her book to "My Friend Ernst Mayr."

The awards that Mayr received include the National Medal of Science, the Balzan Prize, the Sarton Medal of the History of Science Society, the International Prize for Biology, the Loye and Alden Miller Research Award, and the Lewis Thomas Prize for Writing about Science. In 1939 he was elected a Corresponding Member of the Royal Australasian Ornithologists Union. He was awarded the 1946 Leidy Award from the Academy of Natural Sciences of Philadelphia. He was awarded the Linnean Society of London's prestigious Darwin-Wallace Medal in 1958 and the Linnaean Society of New York's inaugural Eisenmann Medal in 1983. For his work, Animal Species and Evolution, he was awarded the Daniel Giraud Elliot Medal from the National Academy of Sciences in 1967. Mayr was elected a Foreign Member of the Royal Society (ForMemRS) in 1988. In 1995 he received the Benjamin Franklin Medal for Distinguished Achievement in the Sciences of the American Philosophical Society. Mayr never won a Nobel Prize, but he noted that there is no prize for evolutionary biology and that Darwin would not have received one, either. (In fact, there is no Nobel Prize for biology.) Mayr did win a 1999 Crafoord Prize. It honors basic research in fields that do not qualify for Nobel Prizes and is administered by the same organization as the Nobel Prize.

In many of his writings, Mayr rejected reductionism in evolutionary biology, arguing that evolutionary pressures act on the whole organism, not on single genes, and that genes can have different effects depending on the other genes present. He advocated a study of the whole genome rather than of isolated genes only. After articulating the biological species concept in 1942, Mayr played a central role in the species Problem debate over what was the best species concept. He staunchly defended the biological species concept against the many definitions of "species" that others proposed.

Mayr encouraged his Linnaean Society seminar participants to take up a specific research project of their own. Under Mayr's influence one of them, Joseph Hickey, went on to write A Guide to Birdwatching (1943). Hickey remembered later, "Mayr was our age and invited on all our field trips. The heckling of this German foreigner was tremendous, but he gave tit for tat, and any modern picture of Dr E. Mayr as a very formal person does not square with my memory of the 1930s. He held his own." A group of eight young Birdwatchers from The Bronx later became the Bronx County Bird Club, led by Ludlow Griscom. "Everyone should have a problem" was the way one Bronx County Bird Club member recalled Mayr's refrain.

As a traditionally trained Biologist, Mayr was often highly critical of early mathematical approaches to evolution such as those of J.B.S. Haldane, famously calling such approaches "beanbag genetics" in 1959. He maintained that factors such as reproductive isolation had to be taken into account. In a similar fashion, Mayr was also quite critical of molecular evolutionary studies such as those of Carl Woese. Current molecular studies in evolution and speciation indicate that although allopatric speciation is the norm, there are numerous cases of sympatric speciation in groups with greater mobility (such as birds). The precise mechanisms of sympatric speciation, however, are usually a form of microallopatry enabled by variations in niche occupancy among individuals within a population.

Mayr was an outspoken defender of the scientific method, and one known to sharply critique science on the edge. As a notable Example, in 1995, he criticized the Search for Extra-Terrestrial Intelligence (SETI) as conducted by fellow Harvard professor Paul Horowitz as being a waste of university and student resources, for its inability to address and answer a scientific question. Over 60 eminent Scientists led by Carl Sagan rebutted the criticism.

Mayr joined the faculty of Harvard University in 1953, where he also served as Director of the Museum of Comparative Zoology from 1961 to 1970. He retired in 1975 as emeritus professor of zoology, showered with honors. Following his retirement, he went on to publish more than 200 articles, in a variety of journals—more than some reputable Scientists publish in their entire careers; 14 of his 25 books were published after he was 65. Even as a centenarian, he continued to write books. On his 100th birthday, he was interviewed by Scientific American magazine. Mayr died on 3 February 2005 in his retirement home in Bedford, Massachusetts after a short illness. His wife, Margarete, died in 1990. He was survived by two daughters, five grandchildren and 10 great-grandchildren.