The dominant German manufacturer swept the podium in their 11th win in the last 13 years at the Sarthe circuit. They won last year with a diesel engine.
"It's really superb as when you come here for the first time you don't know how the technology will work out," Audi motorsport head Wolfgang Ullrich told reporters after the 80th edition of the race.
The number one diesel-hybrid R18 - shared by Lotterer, France's Benoit Treluyer and Switzerland's Marcel Faessler - led for most of the race and denied Denmark's Tom Kristensen a ninth win in the number two Audi.
"We arrived better prepared and as we won last year it gave us confidence," said Treluyer. "We kept the same driver line-up and to be on the hybrid project motivated us even more."
Germany's Mike Rockenfeller, a winner for Audi in 2010, wrapped up the 1-2-3 when he crossed the line in third place in a non-hybrid car.
Audi overcame a scare with three hours to go when British driver Allan McNish slid the number two Audi into the barriers at the Porsche Curves only moments after Spaniard Marc Gene plunged his non-hybrid Audi into the tyres at the first chicane.
McNish's car was quickly back on track, with the Scot handing over to Kristensen, but Gene's number three car lost 20 minutes and finished in fifth place with Frenchman Loic Duval at the wheel.
Frenchman Nicolas Prost, son of four-times Formula One champion Alain, split the Audis by claiming fourth place for Rebellion Racing's Toyota-powered Lola B12.
Scotland's Peter Dumbreck and former Formula One drivers David Brabham and Karun Chandhok, the first Indian to race at Le Mans, finished a strong sixth in the JRM team's HPD ARX 03a.
Toyota saw their challenge fade overnight.
They had snatched the lead after five hours of intense racing but their excitement turned to horror when Anthony Davidson suffered a spectacular crash.
The Briton's Toyota tagged a Ferrari and took off, twisting 360 degrees in the air before plunging into the tyre barriers at Mulsanne Corner.
Davidson, who clambered out of the car before gesticulating for help from the medical team, will stay in hospital until Wednesday after fracturing two vertebrae in the incident.
"Well that was a big one! Feeling a bit sore today, but generally happy to be alive," the 33-year-old, whose injuries could take three months to heal fully, said on Twitter.
Toyota later retired their other car when the engine failed.
"It was a real disappointment to end the race early; our dream was to see the chequered flag," said Kazuki Nakajima, who was the last Toyota driver out on track.
Despite failing to finish, Toyota impressed after returning to sportscar racing for the first time in 13 years with a petrol-electric hybrid car.
Before his exit, Nakajima ended the run of the experimental Nissan DeltaWing overnight when the Toyota pushed it off the road and into the barriers at the exit of the Porsche Curves.
The sleek car, reminiscent of a 'Batmobile', had been invited to race at Le Mans to showcase new technologies, including a smaller engine and four-inch front wheels.
"It hurt last night and it hasn't got any easier," said Marino Franchitti, younger brother of Indy 500 winner Dario and a cousin of Formula One racer Paul di Resta.
"But I'm very proud to show that this car works and what the future of motorsport could be. I hope this is only the beginning of this car."
Ex-Formula One driver and 1990 Le Mans winner Martin Brundle paired up with son Alex for the first time and Spanish gamer-turned-racer Lucas Ordonez in a Zytek and finished 15th.
Final standings after 24 hours:
1. #1 Audi Lotterer/Fassler/Treluyer 378
2. #2 Audi McNish/Capello/Kristensen 377 + 1 Lap
3. #4 Audi Jarvis/Bonanomi/Rockenfeller 375 + 3 Laps
4. #12 Lola Prost/Heidfeld/Jani 367 + 11 Laps
5. #3 Audi Gene/Dumas/Duval 366 + 12 Laps
6. #22 HPD Brabham/Dumbreck/Chandhok 357 + 21 Laps
7. #44 HPD Potolicchio/Dalziel/K-Smith 354 + 24 Laps
8. #46 Oreca Thiriet/Beche/Tinseau 353 + 25 Laps
9. #49 Oreca Perez Companc/Kaffer/Ayari 352 + 26 Laps
10. #26 Oreca Ragues/Panciatici/Rusinov 351 + 27 Laps