The National Museum of Health and Medicine was established during the Civil War as the Army Medical Museum, a center for the collection of specimens for research in military medicine and surgery. In 1862, Surgeon General William Hammond directed medical officers in the field to collect "specimens of morbid anatomy together with projectiles and foreign bodies removed" and to forward them to the newly founded museum for study. The Museum's first curator, John Brinton, visited mid-Atlantic battlefields and solicited contributions from doctors throughout the Union Army. During and after the war, Museum staff took pictures of wounded soldiers showing effects of gunshot wounds as well as results of amputations and other surgical procedures. The information collected was compiled into six volumes of The Medical and Surgical History of the War of the Rebellion, published between 1870 and 1883.

During the late 19th and early 20th centuries, Museum staff engaged in various types of medical research. They pioneered in photomicrographic techniques, established a library and cataloging system which later formed the basis for the National Library of Medicine, and led the Museum into research on infectious diseases while discovering the cause of yellow fever. They contributed to research on vaccinations for typhoid fever, and during World War I, Museum staff were involved in vaccinations and health education campaigns, including major efforts to combat sexually-transmissible diseases.

By World War II, research at the Museum focused increasingly on pathology; in 1946 the Museum became a division of the new Army Institute of Pathology (AIP), which became the Armed Forces Institute of Pathology (AFIP) in 1949. The Museum's library and part of its archives were transferred to the National Library of Medicine when it was created in 1956. The Army Medical Museum became the Medical Museum of the AFIP in 1949, the Armed Forces Medical Museum in 1974, and the National Museum of Health and Medicine in 1989.