Despoina Stylianopoulou studied vocals at the Hellenic Conservatory and theater at the Dimitris Rontiris Drama School, from which she graduated in 1959. She made her film debut in 1960, in the film Stranger in the Night, and since then she has established herself in comedic roles. Her most common role was that of a provincial housemaid who made grammatical errors. She also played the common girl in non-leading roles.

Filmography