The American closed out the victory with a backhand winner, then collapsed to his back as he tossed his racket in jubilation and relief.
The final score of the first-round match read: 6-4, 3-6, 6-7 (7), 7-6 (3), 70-68.
The match totalled 183 games and lasted 11 hours, 5 minutes over three days. The fifth set alone lasted 8 hours, 11 minutes.
The match lasted so long it was suspended because of darkness two nights in a row. Play resumed Thursday at 59-all and continued for more than an hour before Isner won.
The American finished with 112 aces, and Mahut, of France, had 103. There were only three service breaks in the match, the last coming on the final point.
With Mahut serving at 68-69, Isner hit a forehand winner for the first break-point chance of the day. Then he cracked the backhand winner for the victory.
The finish attracted an overflow crowd on cozy Court 18. Absent was Queen Elizabeth II, who had already departed the All England Club following her first visit to Wimbledon since 1977. She watched Briton Andy Murray win his match on Centre Court.
Isner won a match that was by far the longest in the sport's history in terms of games or time. The previous longest match took 6 hours, 33 minutes at the 2004 French Open.
Four times on Wednesday, Isner was one point from victory, but Mahut saved each match point.
The crowd roared when the players walked on the court Thursday, and Isner smiled and waved. When the action resumed, both players continued to dominate with their serves. Mahut held at love five times.
But in the final game, the Frenchman committed an unforced error and netted a drop shot on consecutive points, opening the door for Isner.
With the queen watching from the Royal Box, Murray defeated Jarkko Nieminen of Finland, 6-3, 6-4, 6-2. Murray, seeded fourth, is trying to become the first British player to win Wimbledon since the queen watched Virginia Wade win the women's final 33 years ago.
Shortly after the queen took her seat in the front row, Murray and Nieminen walked onto the court. They turned toward her and simultaneously bowed as the crowd roared.
Murray, the only British player left in singles, might have been a bit nervous at the start. He faced four break points in the opening game but erased them all, then pulled away from there.
The queen joined the applause when Murray closed out the win. Both players again bowed as they left the court, and they then met with the queen on a balcony overlooking the club's outer courts before she departed.
The queen emerged from a car near the club's practice courts an hour before the day's first matches, and walked toward Centre Court along a walkway lined with spectators. When she reached the members' lawn, she met several players, including Roger Federer, Venus and Serena Williams and Andy Roddick, and former Wimbledon champions Martina Navratilova and Billie Jean King.
"She said, 'Oh, you've been coming here a long time,'" King said. "And I said, 'Yes, 49 years in a row,' and she goes, 'Oh, that's wonderful.' ... It was really an honor and a big thrill for me, because it is on my bucket list."
Serena Williams greeted the queen with the curtsy she had been practicing. Roddick and Federer bowed. Spectators cheered as the queen then walked across a bridge to the clubhouse for lunch.
After she left Wimbledon, No. 3-seeded Caroline Wozniacki defeated Chang Kai-Chen on Centre Court, 6-4, 6-3. No. 7 Agnieszka Radwanska defeated Alberta Brianti 6-2, 6-0, No. 10 Flavia Pennetta swept Monica Niculescu 6-1, 6-1, and No. 14 Victoria Azarenka beat Bojana Jovanovski 6-1, 6-4.