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Audi's win in the wet and greasy 76th Le Mans 24-hour classic was the German carmaker's eighth in 10 starts.
The result puts Audi in third place since the event began in 1923, behind winning marque Porsche and one win short of Ferrari.
The race has been cancelled twice: once in 1936 and from 1940 to 1948, during and after World War II.
Audi continues to be the only carmaker to have won with a diesel-powered vehicle. Its main competitor, Peugeot, also ran diesel cars this year, finishing second, third and fifth.
The win was the Audi R10 TDI car's 29th overall race, the 16th victory for the company's diesel-powered racers - and the first win using second-generation biofuel.
The victorious R10 - with drivers Dindo Capello, Allan McNish and Tom Kristensen - covered 5192.649km (381 laps) at an average speed of 216.300km/h, the fifth-quickest in the event's history and the fastest since the Hunaudieres straight was slowed by adding two chicanes in 1990.
The fastest race lap was set by Alexandre Premat in the fourth-placed Audi R10. His time of 3m 23.939s (240.58km/h) was 3.23.7 seconds less than McNish's fastest lap last year.
But McNish owns the all-time lap record - a warm-up run this year of 3m 23.319s (241.317km/h).
He said: "I didn't take risks at the start. The track was treacherous in the rain, like driving on ice.
"Driving in those conditions for over three hours required 100 per cent concentration. If you made the slightest mistake, you didn't just go wide, you would have crashed off and out of the race."
For the fifth time in Le Mans history, and for the first time in 20 years, a vehicle numbered 2 won the event.
During the race, four different cars were running at the front, with the lead changing 25 times altogether. The Audi R10 TDI that ultimately won the race led the field for 178 of the 381 laps - more than any other car.
Of the 55 vehicles that had started from the grid, 19 retired. Audi Sport Team Joest finished the race with all three Audi R10 TDI cars. The winning R10 TDI spent just 31m 56s in the pits. The vehicle pitted 32 times and, thanks to the fuel-efficient TDI engine, had four fewer pit stops than the second-placed Peugeot.
None of the stops was unscheduled. Twenty tyres were changed. Audi says the winning car did not encounter a single technical problem.
One tank of biodiesel in the R10 was enough for 12 laps, one more than rivals Peugeot. On average, the TDi engine swallowed 45.56 litres/100km - or 6mpg.
The winning car had only eight driver changes. McNish had the longest stint: from 6:11am to 9:31am, or 3 hours and 20 minutes.
It was Kristensen's eighth victory at Le Mans, McNish's second, and Capello's third. But this year's win was their first as a team.
Their average age was 40.3 years. Capello turned 44 three days after the race, Kristensen turns 41 on July 7, McNish was 39 last December.
The race was the 26th appearance of a German carmaker, the 50th of an open cockpit sportscar and the 17th for Michelin, Audi's tyre partner, not beaten at Le Mans since 1998.
