Favino was born in Rome, Italy, to Apulian parents. He has appeared in more than forty European films and television series since the early 1990s, including Gabriele Muccino's The Last Kiss, Gianni Amelio's The Keys to the House, Giuseppe Tornatore's The Unknown Woman and Ferzan Özpetek's Saturn in Opposition. In 1999 he starred in the HBO film Excellent Cadavers, an adaptation of Alexander Stille's novel of the same name. In 2006 he received the David di Donatello award - the Italian equivalent of the Oscar - for his role in the crime film Romanzo Criminale, directed by Michele Placido.

In 2006 he portrayed Christopher Columbus in Twentieth Century Fox's Night at the Museum. In 2008 he played General Glozelle, the leader of Miraz's Telmarine troops in The Chronicles of Narnia: Prince Caspian. He has worked with American Director Ron Howard twice: in the 2009 film Angels & Demons, the adaptation of the novel of the same name by Dan Brown, in which he played the role of Inspector Ernesto Olivetti, and in the 2013 film Rush, in which he played the role of Formula One racing car driver Clay Regazzoni.

Other significant roles include the partisan leader Peppi Grotta in Spike Lee's Miracle at St. Anna in 2008, the sadistic riot control force policeman Cobra in Stefano Sollima's ACAB – All Cops Are Bastards in 2012, the anarchist Giuseppe Pinelli in Marco Tullio Giordana's Piazza Fontana, and the W.H.O. Doctor in Marc Forster's apocalyptic-horror World War Z (2013).

In February 2018, he co-hosted the 68th edition of the Sanremo Music Festival, alongside Claudio Baglioni and Michelle Hunziker.