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After several guest roles on television, she became recognized for her portrayal of Isabella Braña on the CBS daytime soap opera The Young and the Restless (2001–2003). She has appeared in the films The Sentinel (2006), Over Her Dead Body (2008), For Greater Glory (2012), Frontera (2014), Lowriders (2016), and Overboard (2018), winning an Imagen Award for the latter. She guest-starred on the Hulu series Only Murders in the Building (2024), earning her a third Screen Actors Guild Award for Outstanding Performance by an Ensemble in a Comedy Series.

In 2005, she founded UnbeliEVAble Entertainment, a film and television production company. In 2023, the company was acquired by Hyphenate Media Group, a production company co-founded by Longoria and Cris Abrego. She also executive produced the Lifetime television series Devious Maids (2013–2016), the short-lived NBC sitcom Telenovela (2015–2016) in which she starred, and the films The Harvest (2013) and Food Chains (2014). Her directorial ventures include the television series Ashley Garcia: Genius in Love (2020), for which she was nominated at the Daytime Creative Arts Emmys, and the Oscar-nominated film Flamin' Hot (2023).

She has appeared in several advertising campaigns, including with L'Oréal, New York & Co, Pepsi, and launched her own fashion and perfume brand in 2017. Her other business ventures include opening the restaurant Beso in 2008, investing in businesses and films such as John Wick (2014), and publishing a cookbook.