When D'Souza was offered a role in Tujhe Meri Kasam, initially she turned it down, as she was not keen to pursue a career in acting. But the crew insisted and kept contacting her for two months, and she agreed when she saw the Telugu version of the movie.Tamil director, S. Shankar, was impressed with her performance in the Parker Pen commercial and decided to cast her in a leading role in his 2003 Tamil film Boys. D'Souza was selected among 300 girls, who had auditioned for the movie. She signed three movies simultaneously in three different languages, Tujhe Meri Kasam (Hindi), Boys (Tamil), and Satyam (Telugu).
In 2004, D'Souza appeared in her second Bollywood movie, Masti. The comedy focuses on three close friends who reunite after three years, but are now married and are being harassed by their wives. D'Souza portrayed the character of one of the wives.Taran Adarsh was complimentary of D'Souza's role, saying, "Amongst the wives, Genelia is the best, Genelia looks the stern and demanding wife and is sure to be noticed.The film fared well at the UK box office.The same year, she appeared in two Telugu movies Samba,and Sye,both succeeding at the box-office.After appearing in her first Telugu movie in 2005, Naa Alludu, she starred in the Tamil romantic entertainer Sachein.A review in The Hindu noted, "Genelia, who hardly made an impression in Boys, makes much impact in Sachein.The movie evoked mixed response from audiences, but was well received with the younger generation.She later appeared in the Telugu patriotic movie Subhash Chandra Bose.
D'Souza has been often tagged in the media as the "bubbly girl", after portraying the role of a young energetic girl in several movies, especially Jaane Tu... Ya Jaane Na (2008). She appeared alongside Shahid Kapoor on the fourth episode of Tere Mere Beach Mein, a celebrity chat show, hosted by Farah Khan. The theme of the show was "Second Innings", since both D'Souza and Kapoor had the same history of failures initially and success later in their acting careers.In June 2010, D'Souza was crowned as the "Brand Ambassador of the Year" at the CNBC Awaaz consumer awards, for endorsing nine brands.She is now the brand ambassador of the soft drink Fanta (replacing Rani Mukherjee), the chocolate Perk (replacing Preity Zinta),Virgin Mobile India (along with Ranbir Kapoor),Fastrack watches and accessories,[ LG mobiles (along with John Abraham and Abhay Deol),Garnier Light fairness cream, Dabur Vatika hair oil, Margo soap, and Spinz deodorant.
She will appear in David Dhawan's film Hook Ya Crook, co-starring John Abraham and Shreyas Talpade.D'Souza is set to appear in It's My Life, which would be a remake of Bomarillu in Hindi with Harman Baweja. Genelia has signed Santosh Sivan's Malayalam film Urumi. The film is about a fictional story happening in the 15th century, about a boy who plots to assassinate the Portuguese explorer Vasco Da Gama. D'Souza plays the role of a Muslim warrior princess Ayesha in the movie.