He is known in Brazil for his appearance on local telenovelas and internationally for his portrayal of Persian King Xerxes in the film 300 (2006) and its sequel 300: Rise of an Empire (2014).

His first major role in a film production came in 2001, with Bicho de Sete Cabeças (Brainstorm) from Brazilian director Laís Bodansky.

After Bicho, his reputation as an actor had been solidified and he was cast as the male lead in Abril Despedaçado (Behind the Sun), one of the nominees for best foreign film in the 2002 Golden Globe Awards.

Canadian director Robert Allan Ackerman signed Santoro for his TV production The Roman Spring of Mrs. Stone after being mesmerized by his performance in Bicho de Sete Cabeças.

Soon after he finished shooting for Mrs. Stone, he received an offer from Columbia Pictures for a part in the blockbuster Charlie's Angels: Full Throttle.

In 2006, Santoro joined the season three cast of the ABC television series Lost as Paulo, a survivor of the Oceanic Flight 815 crash.

Santoro was cast as Persian emperor Xerxes in Zack Snyder's film 300 (2006), based on the Frank Miller comic of the same name.

Santoro played Jesus in the 2016 historical action film Ben-Hur and received personal blessings from Pope Francis for the role.

As the character Marcos, in the award-winning feature film, he emphasized his strong relationship with the city and the reasons that led him to select it as one of the locations for an avant-première: "My mother's entire family, Junqueira Reis, is from Ribeirão Preto.

After the soap opera, Mel was left without the flat provided by the broadcaster and, at Santoro's invitation, she started living in his apartment, in Leblon.