The AMM was established during the American Civil War as a center for the collection of specimens for research in military medicine and surgery. In 1862, 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 AMM's first curator, John H. Brinton, visited mid-Atlantic battlefields and solicited contributions from doctors throughout the Union Army. During and after the war, AMM 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, AMM 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 (NLM), and led the AMM 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, AMM staff were involved in vaccinations and health education campaigns, including major efforts to combat sexually transmissible diseases.
By World War II, research at the AMM focused increasingly on pathology. In 1946 the AMM became a division of the new Army Institute of Pathology (AIP), which became the Armed Forces Institute of Pathology (AFIP) in 1949. The AMM's library and part of its archives were transferred to the National Library of Medicine when that institution was created in 1956. The AMM itself became the Medical Museum of the AFIP in 1949, the Armed Forces Medical Museum in 1974, and finally the NMHM in 1989. During its peak years on the National Mall in the 1960s, every year the Museum saw "as many as 400,000 to 500,000 people coming through". But after its moves to increasingly obscure and out-of-the-way sites, it fell into a period of relative neglect. By the 1990s, it was attracting only between 40,000 and 50,000 visitors a year.
In 1989, C. Everett Koop (in his last year as Surgeon General) commissioned the "National Museum of Health and Medicine Foundation", a private, nonprofit organization to explore avenues for its future development and revitalization, with the aim of ultimately returning its collection to a venue on the National Mall. Proposed was “a site on land that is located east of and adjacent to the Hubert H. Humphrey Building (100 Independence Avenue, Southwest, in the District of Columbia)”. In 1993, a draft bill authored by Sen. Edward Kennedy proposed $21.8 million for moving the existing collection to a new facility to be constructed on that site. That bill, however, was never introduced owing to political difficulties including objections from Constance Breuer—widow of Marcel Breuer, architect of the Humphrey Building—who objected to the view obstruction that would be entailed by the proposed construction. A letter from the Department of Defense to Koop in the mid-1990s, expressed hope that the NMHM exhibits would "one day be provided the appropriate and prominent home they deserve back at the National Mall in the new National Health Museum". But the DoD backed away from contributing to funding a new museum. The Foundation has since been superseded by a new organization, dedicated to creating a new National Health Museum, and which has more ambitious aims and is not dependent on what happens to the existing NMHM.