Reed became a medical officer with the US Army in the 1880s, a time of great advances in medicine due to widespread acceptance of Louis Pasteur's germ theory of disease as well as the methods of studying bacteria developed by Robert Koch. Reed worked closely with George Miller Sternberg, the Army Surgeon General, who was one of the founders of bacteriology.
Yellow fever became a problem for the Army during the Spanish American War, when the disease felled thousands of soldiers in Cuba. In May 1900, Reed, a major, was appointed president of a board "to study infectious diseases in Cuba paying particular attention to yellow fever." This board eventually proved both the transmission by mosiquots and disproved the common belief that yellow fever could be transmitted by clothing and bedding soiled by the body fluids and excrement of yellow fever sufferers - articles known as fomites.
After this work, Reed resumed his position as professor of bacteriology in the Army Medical School, and as professor of pathology and bacteriology at the George Washington University Medical School. His health had been in decline following an appendectomy, and he died of peritonitis. He was buried in Arlington National Cemetery.
Walter Reed General Hospital in Washington, D.C is named after him.