The George Eastman Museum preserves and promotes the art of film in all its forms, from the mainstream to the avant-garde. Founded by the museum’s first curator of film, James Card (1915–2000), the collection now comprises more than 28,000 titles spanning the entire history of international cinema, from the early experiments of Thomas Edison and the Lumière brothers to the present.

In addition to one of the world's finest collections of films from the silent era, the Eastman Museum holds the largest corpus of original Technicolor negatives, including those of Gone With the Wind and The Wizard of Oz; the personal collections of film directors such as Kathryn Bigelow, Ken Burns, Cecil B. DeMille, Norman Jewison, Spike Lee, and Martin Scorsese; and more than four million film stills, posters, and film-related documents.

The Moving Image Stills, Posters, and Paper Collection comprises approximately three million objects, including letters, scripts, musical scores, lobby cards, posters, film stills, and celebrity portraits.

The museum's collection of rare or unique 35mm prints on nitrate stock—the flammable film stock used from the beginning of motion pictures through the middle of the twentieth century—is preserved in the Louis B. Mayer Conservation Center, where films are kept at optimal conditions of temperature and humidity.

The Eastman Museum regularly screens films from its collection in the 500-seat Dryden Theatre, the museum's primary exhibition space for moving images.