The American closed out the victory with a backhand passing shot, 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.
"When you come out and play a match like this, in an atmosphere like this, you don't feel tired really out there," Isner said, "even though that's exactly what we both were."
The match totaled 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 20 games and 65 minutes before Isner won.
In the big Grand Slam tennis tournaments like Wimbledon, there's no quick tie-breaker to decide the match, reports CBS News correspondent Mark Phillips.
It was by far the longest match 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.
"We played the greatest match ever in the greatest place to play tennis," Mahut said. "John deserved to win. He just served unbelievable."
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.
Top-ranked Rafael Nadal, meanwhile, rallied in the second round to beat Robin Haase 5-7, 6-2, 3-6, 6-0, 6-3. Nadal, the 2008 champion who is seeded No. 2 behind Roger Federer, won all 20 points on his serve in the final set and improved to 13-3 in five-set matches.
Other winners included Maria Sharapova, Caroline Wozniacki and Robin Soderling. But those results - and even the queen's visit - were overshadowed by a match that was by far the longest in the sport's history in terms of games and time.
Isner finished with 112 aces, and Mahut, of France, had 103, with both totals eclipsing the sport's previous high of 78. There were only three service breaks in the match, the last coming on the final point. They combined to hold for 168 games in a row.
"It stinks someone had to lose," Isner said. "But to be able to share this day with him was an absolute honor. I wish him nothing but the best, and maybe I'll see him somewhere down the road, and it won't go 70-68."
The crowd roared Isner and Mahut walked on the court Thursday. Mahut wore an intense expression, while Isner smiled and waved. The electronic scoreboard was again working after it froze and then went blank as the fifth set approached 100 games Wednesday.
When the match resumed and Isner held after reaching deuce, the chair umpire announced the score.
"Isner leads 60 games to 59, final set," he said. The crowd laughed.
Both players continued to dominate with their serves. Mahut, always trailing when he served, held at love five times, and appeared to be the fresher player.
He hit a leaping overhead to end one exchange and skipped to the baseline following a changeover, while the 6-foot-9 Isner moved deliberately between points. But in the 183rd and final game, the Frenchman sailed a forehand long and netted a drop shot on consecutive points to fall behind 15-30.
Isner hit a forehand winner for the first breakpoint chance of the day. Then he cracked the backhand winner for the victory - just the third service break of the match, and the only one in the fifth set.
The shot came at 4:48 p.m. - nearly 48 hours after the match started. The crowd gave both players a standing ovation that lasted long after the bear hug, and as Mahut sat in his chair with a towel draped around his head, Isner pointed at him and joined the applause.
Seeded 23rd, Isner will be back on court for a fourth day in a row Friday to play a second-round match.
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.
Over on Court 1, Sharapova advanced to the third round by beating Ioana Raluca Olaru 6-1, 6-4. Seeded 16th, Sharapova won 20 of 23 points at the net.
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 Federer, Venus and Serena Williams and Andy Roddick, and former Wimbledon champions Martina Navratilova and Billie Jean King.
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.
Records set in John Isner's 6-4, 3-6, 6-7 (7), 7-6 (3), 70-68 victory over Nicolas Mahut:
Longest match - 11 hours, 5 minutes
Longest set - 8 hours, 11 minutes
Most games in a match - 183
Most games in a set - 138
Most aces - 215 (Isner 112, Mahut 103)