D'Souza was born to Jeanette D'Souza, formerly secretary to Managing Director with a pharma MNC, and Neil D'Souza, a senior official with Tata Consultancy Services (TCS). Her family hails from the Mangalorean Catholic community, a prominent Christian community in Bombay. Her nick name is Geenu. D'Souza studied at the Apostolic Carmel High School in Bandra and later joined St. Andrews College (Bandra) to pursue her Bachelor's Degree of Management Studies. 
She was also a State level athlete and a National level Football player.
D'Souza made her debut in Bollywood in 2003. Her first Hindi film was Tujhe Meri Kasam opposite Ritesh Deshmukh. She left Bollywood for a while to concentrate on South Indian films. She first hit the lime-light with a Parker Pen commercial with Amitabh Bachchan and further did a Fair and Lovely 2003 Cricket World Cup ad with Krishnamachari Srikkanth. She was also a part of Mani Ratnam's stage show, Netru, Indru, Naalai. The Tamil director, Shankar, was very much impressed with her performance in both the ads and cast her in a leading role in his Tamil film Boys in 2003 which was a huge success.
She made her Telugu debut with Sathyam opposite Sumanth, which went on to become a blockbuster. Subsequently, she acted in several films which raised her profile in south India. She acted in successful telugu films like Happy, and Sye. She followed it up with a pairing with actor Venkatesh in Subhash Chandra Bose which met with disappointment at the box-office.
Her second Hindi movie was Masti and was also opposite Ritesh. This film was a moderate success.
She then played the role of Haasini in the Telugu film, Bommarillu, which also starred Siddharth Narayan in the lead. This movie was one of the biggest hits in her career and earned her critical acclaim and wide recognition. She won the Filmfare Award as the Best Actress for her performance, besides other awards. Her character of Haasini earned rave reviews and this made her the favourite choice for that role in the remakes of Bommarillu in other languages. It was remade in Tamil as Santhosh Subramaniam in which Genelia was cast as heroine again. The film also turned out to be rather successful as its predecessor. Her debut film in Kannada Satya In Love grossed 4 crores in its opening week thus setting a new record in the Kannada film industry.
She once again appeared in Bollywood in Mere Baap Pehle Aap alongside Akshaye Khanna, which was moderately successful. Her final breakthrough in Bollywood was through the blockbuster, Jaane Tu Ya Jaane Na, where she is the heroine in the role of Aditi Mahant aka "Meow". Her role has been widely admired for her sweetness and freshness, and new style of acting. The movie has debutant Imran Khan, Aamir Khan's nephew, as the hero. The movie released on July 4, 2008 and was a success across India and overseas. Her career in south India also continues with she reigning as a successful star in Telugu film industry. Her latest Telugu film, Ready, in which she was paired opposite Ram, was a super hit. Her next film, It's My Life, which is again a remake of Bomarillu with Harman Baweja, is to be released in 2009.
She will also act in David Dhawan's next film, a cricket film called Hook Ya Crook, opposite John Abraham and Shreyas Talpade for the first time; the film will be released in 2009. She also has the other film on her hand which is to released by mid of 2009 which is titled Life Partner with Fardeen Khan. She is now the brand ambassador of the new Fanta replacing Rani Mukherjee. She is also now the brand ambassador of chocolate Perk replacing Preity Zinta.
Check her out in her new movie releasing in 2010 "Chance Pe Dance" with Heartthrob Shahid Kapur