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Born in Palermo, Guadagnino spent part of his childhood in Ethiopia. However, the family returned to Italy to escape the Ethiopian Civil War. Guadagnino began his career directing short films and documentaries. He made his feature-film debut with The Protagonists (1999), the first of his many collaborations with actress Tilda Swinton. His follow-up, Melissa P. (2005), was a commercial success in Italy but was met with mixed critical reception.

Guadagnino gained further acclaim with his Desire trilogy, which consists of the films I Am Love (2009), A Bigger Splash (2015), and Call Me by Your Name (2017). The latter brought him international recognition. Suspiria (2018), a remake of the 1977 film, was Guadagnino's first foray into the horror genre. It was a box office failure and polarized critics. Guadagnino's following projects were We Are Who We Are (2020), a coming-of-age miniseries for HBO, the romantic horror film Bones and All (2022), the romantic sports film Challengers (2024), and the period romantic drama Queer (2024).

Guadagnino directed several documentaries, including Bertolucci on Bertolucci (2013) and Salvatore: The Shoemaker of Dreams (2020). Aside from filmmaking, he has been involved in the fashion world, directing advertisements for brands like Fendi and Salvatore Ferragamo. In 2012, Guadagnino founded the production company Frenesy Film Company. He also produced Belluscone: A Sicilian Story (2014), The Truffle Hunters (2020), Salvatore: The Shoemaker of Dreams (2020), Holiday, and Enea (2023).