Mandylor was born in Melbourne the son of Greek immigrants from the state of the Peloponnese, Louise (née Mandylaris) from Kalamata and Yannis Theodosopoulos from Tripoli, who is a taxi driver. Mandylor took a version of his mother's maiden name, citing his real name in Greek being too long. He grew up in St Kilda and South Melbourne. He moved to the United States in 1987 where he started taking acting lessons. He was initially unable to find any work acting and had to work any job available.

Mandylor's first major role was in the 1989 film Triumph of the Spirit playing a European Jew, which was filmed at the Auschwitz-Birkenau death camp. He returned to Los Angeles meeting Director Oliver Stone and auditioned and got the role of an Italian count in The Doors (1991). Mandylor landed a leading role in Mobsters playing Mafia boss Frank Costello. He went mentally prepared for the role, telling the Los Angeles Times, "It was interesting that I got that part, because I felt tuned in to Gangsters, it was a genre I'd read a lot. When I was 13, I got a job as a dishwasher in a Melbourne nightclub and saw everything there, real Gangsters, and had met characters who were dangerous people from an underground world." In 1992 he had a lead role on the CBS television drama, Picket Fences, playing officer Kenny Lacos until the show ended in 1996. Mandylor also had a lead role in the Saw films as Detective Mark Hoffman.

Mandylor played professional soccer in Europe until suffering shin splints. In 1991, he was chosen by People as one of the "50 Most Beautiful People in the world". Mandylor married Talisa Soto in May 1997; they divorced in 2000. Mandylor married Victoria Ramos in the fall of 2013. His brother, Louis Mandylor, is also an actor.