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Born in Guatemala, Isaac moved with his family to the United States as an infant. As a teenager, he joined a punk band, acted in plays and made his film debut in a minor role. A graduate of the Juilliard School, Isaac was a character actor in films for much of the 2000s. His first major role was that of Joseph in the biblical drama The Nativity Story (2006), and he won an AACTA Award for Best Actor in a Supporting Role for portraying political leader José Ramos-Horta in the Australian film Balibo (2009). After gaining recognition for playing supporting parts in Robin Hood (2010) and Drive (2011), Isaac had his breakthrough with the eponymous role of a singer in the musical drama Inside Llewyn Davis (2013), which earned him a Golden Globe nomination.

Isaac's career progressed with leading roles in the crime drama A Most Violent Year (2014), the thriller Ex Machina (2015) and the superhero film X-Men: Apocalypse (2016). He became a global star with the role of Poe Dameron in the Star Wars sequel trilogy (2015–2019). Isaac starred in the historical drama Operation Finale (2018)—which marked his first venture into production—the science fiction films Annihilation (2018), Dune (2021), and Frankenstein (2025), the crime drama The Card Counter (2021), and the animated superhero film Spider-Man: Across the Spider-Verse (2023).

On television, Isaac was the lead in three miniseries: Show Me a Hero (2015), in which his portrayal of Nick Wasicsko won him a Golden Globe Award, Scenes from a Marriage (2021), and the Marvel Cinematic Universe's Moon Knight (2022). His stage work includes title roles in Romeo and Juliet (2007), Hamlet (2017) and The Sign in Sidney Brustein's Window (2023).