Joseph Gregor grew up in Czernowitz as the son of the city architect Josef Gregor. He came to Vienna in 1907 to study art history, German and music. He obtained his doctorate in 1911 with a musicological thesis, but his main interest had always been the theatre. After studying at the Wiener Hofoper, the Deutsches Theater in Berlin under the direction of Max Reinhardt and the University of Leipzig under Albert Köster, he returned in 1912 to Czernowitz to become a university music lecturer. He served during the First World War in the Tiroler Kaiserjäger regiment and as a Kriegswirtschaftskommissär (War Economy Commissar) but remained resident in Vienna. In 1918 he embarked on a career at the National Library, where he was entrusted after just two years with the establishment of a theatre department, whose head he became in 1922. He was also head of the Bundestheatermuseum (Federal Theatre Museum) founded in 1931 and was awarded the title of professor in 1933 and a few years later the rank of Oberstaatsbibliothekar (senior state librarian). When he applied as a writer for admission to the Reichsschrifttumskammer (Reich Literature Chamber) after the annexation of Austria to the German Reich, opinions as to his political reliability were split. He was thought to be sympathetic to the National Socialist movement, but as editor of the magazine Die Theater der Welt in 1936/37 he was suspected of being friendly with Jews and internationalists. He had already had to passed on his role as editor in 1937 to Karl Niessen, head of the Institute of Theatre Studies at the University of Cologne. The mistrust of the Ministry of Propaganda in Berlin combined with the efforts of his rival Niessen almost resulted in the theatre collection being moved to Berlin or Cologne and Gregor being removed from office. Thanks to Gregor's contacts and those of his staunch supporter Paul Heigl, director general of the National Library, the institute was not transferred and Gregor was left in peace in spite of the unfavourable political assessment by the NSDAP. He was ultimately able to enlarge the theatre collection during the Nazi era through donations, purchases and also Aryanization. Gregor retired at the end of 1953 and in subsequent years he lectured in the history of theatre at the Akademie der bildenden Künste (Academy of Fine Arts) and the Akademie für Musik und darstellende Kunst (Academy of Music and Performing Arts) in Vienna. During his lifetime he published an impressive number of articles about the history of theatre.

Since 1998 objects that Gregor had acquired during the Nazi era have been successively restituted.