Born in Mumbai into a Mangalorean Catholic family, Genelia was raised as a Roman Catholic in the Bandra suburb of Mumbai. Her mother tongue is Konkani. Her mother Jeanette D'Souza was formerly a managing director of the Pharma Multinational corporation (MNC), who left her job in 2004 to help Genelia with her career. Her father Neil D'Souza, is a senior official with Tata Consultancy Services (TCS). She also has a younger brother, Nigel D'Souza, who works with the Bombay Stock Exchange. According to Genelia, her name means "rare" or "unique", and is a portmanteau of Jeanette and Neil, her mother and father's names. She is also often informally referred to as Geenu, her nickname.Genelia studied at the Apostolic Carmel High School in Bandra and later joined St. Andrew's College in Bandra to pursue her bachelor's degree of Management Studies. She completed her degree while shooting for her first film, Tujhe Meri Kasam in 2003 and initially thought that an MNC job would suit her. She liked sports and studies in college, and was a state level athlete, sprinter, and a national level football player.
Genelia did her first modeling assignment at the age of 15, the result of being spotted as the bridesmaid at a wedding. She was selected for the Parker Pen commercial with Amitabh Bachchan, just two days before her exams, and had to shoot the next day. Initially she refused, because of her exam the next day, but the director persuaded Genelia to shoot for the commercial. She gained wide attention from the Parker Pen commercial with Amitabh Bachchan, who said "she was good, and her expressions were spontaneous". She further did a Fair and Lovely 2003 Cricket World Cup advertisement with cricketer Krishnamachari Srikkanth.
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. In an interview with The Times of India, she comments, "I keep a Novena every Wednesday at St. Michael's Church in Mahim." In an interview with Daily News and Analysis, she said that "My communication with God is conversational, I'm God's favourite child; I believe that God has always been kind to me." Her mother tongue is Konkani. Tabloids repeatedly linked D'Souza romantically with Ritesh Deshmukh, ever since they starred together in their debut film Tujhe Meri Kasam in 2003. They were reportedly ready to get engaged, but Ritesh's father, the then-Maharashtra Chief Minister, Vilasrao Deshmukh did not agree. D'Souza later denied any rumors of a relationship with Deshmukh, and responds that she had just friendly relations with him. However, the couple eventually got married on 3 February 2012, according to Marathi marriage traditions in a Hindu wedding ceremony. She got married in a Roman Catholic ceremony in Bandra, the following day. The couple's first child, a son named Riaan, was born on 25 November 2014. Their second son was born on 1 June 2016.