Enders was a student of English literature at Harvard University (M.A., 1922) before he turned to bacterial studies there (Ph.D., 1930). His early researches contributed new and basic knowledge to problems of tuberculosis, pneumococcal infections, and resistance to bacterial diseases. In 1929 he joined the Harvard faculty as an assistant in the department of bacteriology and immunology, later advancing to assistant professor (1935) and associate professor (1942) in the university's medical school.

In World War II he was a civilian consultant on infectious diseases to the U.S. War Department. From 1945 to 1949 he served the U.S. Army in a like capacity, with particular work on the mumps virus and rickettsial diseases. During this period Enders, with his coworkers Weller and Robbins, began research into new methods of producing in quantity the virus of poliomyelitis. Until that time the only effective method of growing the virus had been in the nerve tissue of living monkeys, and the vaccine thus produced had been proved dangerous to humans. The Enders–Weller–Robbins method of production, achieved in test tubes using cultures of nonnerve tissue from human embryos and monkeys, led to the development of the Salk vaccine for polio in 1954. Similarly, their production in the late 1950s of a vaccine against the measles led to the development of a licensed vaccine in the United States in 1963. Much of Enders' research on viruses was conducted at the Children's Hospital in Boston, where he had established a laboratory in 1946.