The Archives was founded by Jonas Mekas, Stan Brakhage, P. Adams Sitney and Peter Kubelka and opened on November 30th, 1970. Today it keeps ca. 11,000 films and 3,000 videotapes and is thus one of the largest archives of avant-garde and experimental cinema in the world. The collection which focuses on American filmmakers also holds many commercial and industrial negatives deposited at the institution by insolvent films laboratories.
In 2005, the Archives released Unseen Cinema: Early American Avant Garde Film 1894-1941, a DVD retrospective of early experimental film. That same year, the Archives was among 406 New York City arts and social service institutions to receive part of a $20 million grant from the Carnegie Corporation, which was made possible through a donation by New York City mayor Michael Bloomberg.

From 2004 through 2006, members of the Anthology Film Archives community organized LAB HD, an ambient televisionchannel that broadcast in high-definition nationally in the USA. During this period LAB HD produced dozens of experimental and avant-garde films in its facilities.
In 2007, it was among over 530 New York City arts and social service institutions to receive part of a $20 million grant from the Carnegie Corporation, which was made possible through a donation by New York City mayor Michael Bloomberg.
In the 2004 movie Spider-Man 2, the building was used as the exterior of Doctor Octopus' laboratory.