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Matthew Del Negro nasceu em Mount Kisco, Nova York, como o caçula de três filhos. Ele cresceu no Condado de Westchester. Ele se formou no Boston College, onde jogou lacrosse da primeira divisão.
Depois de se formar, Del Negro começou a estudar atuação e, depois de aparecer em vários comerciais menores e filmes independentes, foi escalado como Brian Cammarata em Os Sopranos. Isso levou a aparições em outras séries de televisão, como The West Wing, Law & Order, Scandal, Stargate: Atlantis, Las Vegas, Beautiful People, CSI: Crime Scene Investigation, NCIS, Joan of Arcadia, Happy Endings, Rizzoli and Isles , Lie to Me, Teen Wolf e United States of Tara. Ele atualmente está em Goliath no Amazon Prime.
Suas participações em filmes incluem Chelsea Walls, Ira e Abby, Trailer Park of Terror, Saving Lincoln e o próximo filme Don't Mess with Texas. Ele também foi escalado para várias produções teatrais. Ele forneceu a voz da Ops Com no videogame SOCOM 4: SEALs da Marinha dos EUA de 2011. Outros trabalhos de videogame incluem L.A. Noire e Mass Effect 3.
Em 2019, Del Negro começou um papel recorrente na série de drama policial da Showtime City on a Hill; ele foi promovido a regular na segunda temporada da série.
Atualmente ele dá aulas de atuação em Santa Monica.
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