[4] After gaining attention in a Parker Pen commercial with Amitabh Bachchan, D'Souza began her acting career with the Hindi film Tujhe Meri Kasam in 2003.

D'Souza went onto appear in such commercially successful films as Masti (2004), Sye (2004), Sachein (2005), Happy (2006), Dhee (2007), Ready (2008), Katha (2009), Urumi (2011), Force (2011), Velayudham (2011) and Tere Naal Love Ho Gaya (2012).

The movie, though noted for its vulgar sexual content, was a box–office success,[30] and subsequently she started receiving Telugu film offers.

[42] She later appeared in the Telugu patriotic film Subhash Chandra Bose alongside Venkatesh, a box office failure.

[45] She then portrayed the role of Haasini, a vibrant, effervescent and a happy–go–lucky young girl, in the 2006 Telugu romantic comedy Bommarillu, reuniting with Siddharth.

"[51] Following the success of Bommarillu, D'Souza played the role of the daughter of a local don, in the Tamil gangster film Chennai Kadhal alongside Boys co-star Bharath.

"[52] Shortly afterward, she played the role of the sister of a notorious gangster in the blockbuster Telugu film Dhee alongside Vishnu Manchu, set against a gang war backdrop.

[55] The following year, D'Souza appeared in the Telugu romantic thriller Mr. Medhavi with Raja, in which she portrayed the role of a student from Canada.

"[60] In June 2008, following a nearly 5–year gap from Hindi cinema, she appeared in Mere Baap Pehle Aap alongside Akshaye Khanna,[61] which failed to make profit at the box–office.

[62] Sukanya Verma noted, "besides her apparent cuteness, brings in tons of freshness and traits to the youthful characters she chooses to play",[63] while Sonia Chopra criticised her stating, "D'Souza is sprightly but has a standard two–three expressions bank in this film.

[68] Her role was widely admired for her sweetness and freshness, with Raja Sen describing her acting as a "spark that has been missing in Hindi cinema for well over a decade now".

"[73] Her next appearance was in the Telugu thriller Katha with Adith Arun, which was well received,[56] and for which she won the 2009 Nandi Special Jury Award.

[75] In 2010, D'Souza first played a chronographer opposite Shahid Kapoor in Chance Pe Dance, which received poor reviews from critics.

[83] In her first release of 2011, D'Souza made her Malayalam film debut, starring as the Muslim warrior princess Arackal Ayesha, in Urumi alongside Prithviraj Sukumaran.

[84][85] The film is about a fictional story happening in Calicut, Kerala during the 15th century, about a boy who plots to assassinate the Portuguese explorer Vasco Da Gama.

[86] For her role, she learned horse riding, and spent two weeks of training to use the sword, the short stick, and movements of the Dravidian martial art, Kalarippayattu.

[87][88] It was a major commercial and critical success, with The New Indian Express stating: "Genelia breaks her perpetual imbecility morphing into Ayesha, the warrior princess.

"[89] She next played an independent woman in the Hindi film, Force opposite John Abraham, which received mixed reviews from critics but was a box office success.

[102] In 2020, her much delayed Hindi remake of Bommarillu, It's My Life was released directly on Zee Cinema, where she appeared opposite Harman Baweja.

[110] Nandini Ramnath of Scroll.in noted: "Genelia Deshmukh turn out lovely performance, either as a make-believe couple [with Kaul] or as individual trying to do what is best for Romi.

[113] D'Souza is deeply religious and says that she regularly attends Sunday Mass at St. Anne's Parish (Bandra), and whenever the family is home, a part of their evening is reserved for saying the rosary together.

"[10] Tabloids repeatedly linked D'Souza romantically with Ritesh Deshmukh, ever since they starred together in their debut film Tujhe Meri Kasam in 2003.

[120][121] D'Souza was a part of Tamil director Mani Ratnam's stage show, Netru, Indru, Naalai, an event which seeks to raise funds for The Banyan, a voluntary organisation which rehabilitates homeless women with mental illness in Chennai.

[125] In October 2009, she appeared as a showstopper for jewellery designer Farah Khan Ali on the second season of Housing Development and Infrastructure Limited (HDIL) India Couture Week.

[126][127] On 24 October 2009, D'Souza began hosting Big Switch, a television show based on slum kids to reach a bigger audience.

[132] At the Chennai International Fashion Week (CIFW) in December 2009, she appeared as a showstopper for designer Ishita Singh's spring–summer indigenous collection of 2010.

[139] D'Souza holds a Limca world record of delivering four successful [super hit] films in four different languages: Ready (Telugu), Satya in Love (Kannada), Santosh Subramaniam (Tamil) and Jaane Tu...

[140] D'Souza appeared alongside Shahid Kapoor on the fourth episode of Tere Mere Beach Mein, a celebrity chat show, hosted by Farah Khan.

[131] She is now the brand ambassador of the soft drink Fanta (replacing Rani Mukherjee), the chocolate Perk (replacing Preity Zinta),[143] Virgin Mobile India (along with Ranbir Kapoor),[144] Fastrack watches and accessories,[145] LG mobiles (along with John Abraham and Abhay Deol),[146] Garnier Light fairness cream,[147] Dabur Vatika hair oil, Margo soap,[131] and Spinz deodorant.

Again, following the reports of Ritesh Deshmukh and D'Souza's marriage plans, the priest landed up at producer Vipul Amrutlal Shah's office to register a complaint.