Assante has played a number of tough hero characters, such as his starring role as private eye Mike Hammer in the film I, the Jury (1982) as well as Mafia gangsters such as Michael Moretti in Sidney Sheldon's Rage of Angels.

In 1990 his role as Roberto Texador in Sidney Lumet's film Q&A garnered him a Golden Globe nomination, and the following year he portrayed Bugsy Siegel in Neil Simon's The Marrying Man (1991).

In addition to mafia and "tough-guy" roles, Assante has appeared in historical dramas, such as Napoleon Bonaparte in 1987's Napoleon and Josephine: A Love Story opposite Jacqueline Bisset, the heroic Odysseus in The Odyssey, Friedrich Nietzsche in the film adaptation of Irving Yalom's novel When Nietzsche Wept, and as Sanchez, Queen Isabella's Minister of State, in 1492: Conquest of Paradise.

Other film appearances include one as a Cuban bandleader in The Mambo Kings opposite Antonio Banderas and in the adaptation of the science-fiction story Judge Dredd with Sylvester Stallone, his co-star in Paradise Alley.

Outside of the U.S., he has participated in several film projects and humanitarian endeavors in Romania, Bulgaria, North Macedonia, Serbia, Croatia, Bosnia-Herzegovina, Kazakhstan, and Uzbekistan.