De Villota was born in Madrid into a motorsport family. Her father, Emilio de Villota, was a Formula One driver who competed in the 1970s and 1980s, and her brother Emilio de Villota Jr. also raced in Formula Palmer Audi. Growing up surrounded by racing, she pursued a career across multiple disciplines. Her early results included competing in the 2005 24 Hours of Daytona endurance race, appearances in the World Touring Car Championship, and stints in the ADAC Procar Series.
In August 2009 de Villota joined the Superleague Formula series, signing to drive for the Atlético Madrid entry for the remainder of that season. The Superleague Formula concept paired racing teams with European football clubs, each fielding open-wheel cars in their club's colours. De Villota remained with Atlético Madrid until the series folded in 2011, establishing her as one of the more prominent female single-seater competitors in European motorsport of that period. She also contested the Euroseries 3000 during this phase of her career.
In August 2011 Lotus Renault GP confirmed that de Villota had made her Formula One test debut in a Renault R29 at the Paul Ricard Circuit in France. Her management entered talks with the team about securing a test or third driver role for 2012, and she publicly expressed her interest in continuing with the programme.
On 7 March 2012, Marussia F1 Team announced that de Villota had joined as a test driver, with the prospect of running in the team's car later in the season. The appointment was well received given the rarity of women reaching the upper tier of testing roles in Formula One.
On 3 July 2012, de Villota arrived at Duxford Aerodrome in Cambridgeshire for straight-line aerodynamic testing in the Marussia car — her first time behind the wheel of the vehicle. Shortly before 09:30, at the end of a test run as the car re-entered the service area, it collided with a stationary support truck. A BBC reporter on site estimated the impact speed at between 30 and 40 miles per hour.
De Villota was trapped in the car for approximately an hour before being extracted. She was airlifted to Addenbrooke's Hospital in Cambridge with life-threatening head and facial injuries. The team later confirmed that she had lost her right eye. Subsequent medical reports noted the severity of cranial trauma she had sustained.
An internal investigation by Marussia in July 2012 concluded that the car was not a contributing factor in the accident. A report published in 2015 by the UK Health and Safety Executive determined that de Villota had not received full guidance on stopping procedures for the car, and that the vehicle's anti-stall system had activated as she attempted to brake to a standstill, pushing the car forward into the tail-lift of the team's truck.
After 17 days in hospital de Villota was discharged and returned to Spain. She made her first public appearance in October 2012, giving an interview to the Spanish magazine Hola and hosting a press conference. She disclosed that the accident had cost her the senses of smell and taste in addition to her eye, and that she still experienced regular headaches, but she did not rule out a return to racing if she could be granted a licence. She also expressed a wish to campaign for improved safety standards in motorsport.
On 28 July 2013 de Villota married Rodrigo García Millán in Seville. She was preparing to launch her autobiography, La vida es un regalo (Life Is a Gift), in mid-October and was due to speak at a conference in Seville when, on the morning of 11 October 2013 — exactly one year after her first post-accident public appearance — she was found dead in her hotel room at the age of 34. An autopsy confirmed cardiac arrest as the immediate cause. A subsequent forensic examination concluded that her death was a consequence of the neurological injuries sustained in the 2012 accident.
A minute's silence was observed before the 2013 Japanese Grand Prix on 13 October 2013.
The final corner of the Circuito del Jarama leading onto the pit straight was named Curva María de Villota in 2017. The circuit also established an annual Christmas Eve road race in her memory. The Maria de Villota Legacy Foundation, created in 2014, funds treatment for children with genetic neuromuscular illnesses. Formula One driver Carlos Sainz Jr., whom de Villota mentored during his karting career, carries a star on his helmet representing her and was named an official ambassador of her foundation in 2016. In October 2013 de Villota was posthumously awarded the Golden Medal of the Royal Order of Sports Merit by the Spanish government.