Von Praunheim was born as Holger Radtke in Riga, Latvia Central Prison during the German occupation of Latvia in World War II. His biological mother died in 1946 at the psychiatric hospital in Berlin Wittenauer Heilstätten. After his birth, he was given up for adoption. He only knew these facts when his adoptive mother, Gertrud Mischwitzky, told him in 2000. He discovered the fate of his biological mother in 2006 after a lengthy investigation. He documented his quest in the film Two Mothers (2007).

He received the name Holger Mitschwitzky and spent his early years in East Berlin. In 1953, he escaped from East Germany with his family to West Germany, first to the Rhineland moving later to Frankfurt am Main. After Praunheim left the pre-university high school in Frankfurt (Gymnasium), he studied at the Werkkunstschule in Offenbach. He then transferred to the Berlin University of the Arts where he studied fine arts but did not graduate. He initially worked as painter, but eventually opted for a career in filmmaking.

In the late 1960s, he began experimenting in film and creative writing. He made his debut associated with Werner Schroeter with experimental and short movies, like Sisters of the Revolutions (1968) and Samuel Beckett (1969), with which he soon became famous. In the mid sixties, he assumed the stage name “Rosa von Praunheim”, a portmanteau of the Frankfurt quarter Praunheim and German ‘rosa’ for ‘pink’, alluding to the pink triangle that homosexual prisoners had to wear in concentration camps. Praunheim married the actress Carla Aulaulu in 1969. The marriage ended two years later in divorce. During this same period, he also collaborated with Elfi Mikesch in a number of film projects.

Praunheim’s first big feature film was produced in 1970: Die Bettwurst (The Bolsters), a parody of bourgeois marriage. It became a cult movie, which had a sequel in 1973 (Berliner Bettwurst). In 1971 he also caused a stir with his documentary It Is Not the Homosexual Who Is Perverse, But the Society in Which He Lives which led to several gay rights groups being founded and was the beginning of the modern gay liberation movement in Germany, Austria and Switzerland. This movie affected gays in the US, too.

A prolific and controversial filmmaker, Praunheim has centered his directorial efforts in documentaries featuring gay related themes. In the early 1970s he lived for some time in the United States where he made a series of documentaries about post-Stonewall American gay scene. In Army of Lovers or Revolt of the Perverts (1972–1976) he took on the American gay and lesbian movement from the 1950s to 1976. He was also interested in the underground theater in New York City which was the focus of some of his films of this period including Underground and Emigrants (1975). In 1979 Praunheim obtained a German Film Award for Tally Brown, New York, a documentary about the singer and actress Tally Brown.

Back in Berlin, he made feature films such as Red Love (1980) , Our Corpses Are Still Alive (1981), and City of Lost Souls (1983). These films were shown in film festivals worldwide. His feature film, Horror vacui, won the Los Angeles Film Critics Award for best experimental film in 1985. Anita: Dances of Vice (1988) the life story of a scandalous nude dancer in Berlin in the 1920s, earned international attention.

With the eruption of the AIDS epidemic, Praunheim worked in a tetralogy of AIDS themed documentaries. A Virus Knows No Morals (1985), was one of the first feature films about AIDS. The documentaries Positive and Silence = Death, both shot in 1989, deal with aspects of AIDS activism in New York. Fire Under Your Ass (1990) focuses on AIDS in Berlin.

In Germany, Rosa was very vocal in his efforts to educate people about the danger of AIDS and the necessity of practicing Safer Sex. These efforts alienated many gays who came to consider him a moralistic panic-mongerer. He would remain a highly controversial figure in his native country. On 10 December 1991, Praunheim created a scandal in Germany when he outed, among others, the anchorman Alfred Biolek and the comedian Hape Kerkeling in the TV show Explosiv – Der heiße Stuhl as gay. After the show several celebrities had their coming out. In 1999, he made Geisendörfer Medienpreis for Wunderbares Wrodow, a documentary about the people in and around a German village and its castle.

Until 2006, Rosa von Praunheim taught directing at the Film & Television Academy (HFF) “Konrad Wolf” Potsdam-Babelsberg. In 2008, his film Two Mothers was shown at the Tribeca Film Festival and was nominated for “Best documentary”. At the 63rd Berlin International Film Festival, he was awarded with the Berlinale Camera. In 2012, he was awarded with the Grimme-Preis for his documentary Rent Boys. In 2015, he received the Order of Merit of the Federal Republic of Germany. In 2020, he was awarded with the Max-Ophüls-Preis for his life’s work.