In the longest three-set match in Olympic tennis history, fifth seed Tsonga prevailed 6-3 3-6 25-23 in three hours and 57 minutes to reach the last 16. The third set alone lasted three hours.
American Isner and Frenchman Mahut memorably fought each other to a standstill on Wimbledon's grasscourts before Isner won 6-4 3-6 6-7 7-6 70-68 in an astonishing 11 hour five minutes battle.
Tsonga still found the energy to skip around the court kissing his shirt in celebration.
"For me it's good because this is the only way to write my name in the history for the moment," a smiling Tsonga said.
"With Rafa (Nadal), Roger (Federer) and Novak (Djokovic), even Andy (Murray), it's tough to go through big tournaments. So I'm really happy. I hope I will have some more."
Second seed Djokovic, Wimbledon runner-up Murray, three-time Olympic gold medallist Venus Williams and women's top seed Victoria Azarenka were among others advancing to the third round on a busy day at Wimbledon, attended by Prince Albert of Monaco.
Serbian Djokovic, continuing his quest to better his Beijing bronze medal, breezed to a 6-2 6-1 win in less than an hour over American Andy Roddick, whose stars and stripes trainers failed to inspire him.
"Today everything was working perfectly well and I was neutralising his serve by getting a lot of returns back in the court and serving a lot of aces," said Djokovic, who next faces Australian Lleyton Hewitt.
"You're not playing for yourself like you are most of the time throughout the year. This is one of the unique events, unique opportunities to play for your country and the nation is cheering for you."
Briton Murray, who has played both his singles matches under the Centre Court roof due to rain, made light work of Finland's Jarkko Nieminen to book his place in the last 16, winning 6-2 6-4.
To the delight of the home crowd Murray, whose last match against Nieminen at Wimbledon in 2010 was played in front of the Queen, broke his opponent in the first game and again in the fifth to quickly take a 4-1 lead before wrapping up the set.
Nieminen fought back energetically, saving a match point before going head first over the barriers when chasing a backhand in the next game. Murray, leading 5-4, secured victory with an ace.
Third seed Murray will play Marcos Baghdatis, against whom he dropped a set in their third round meeting at Wimbledon, for a place in the quarter-finals after the Cypriot knocked out France's Richard Gasquet 6-4 6-4.
"He will have the upper hand against me, but you know I will just go out there and give it a try, you never know," world number 44 Baghdatis, who kissed the grass court in celebration after his second round win, said of his upcoming clash against Murray.
"I am in the 16's of an Olympic event and that's an incredible thing for me and I hope I will keep going."
Injury-prone Hewitt clinched a surprise win over 13th seed Croatian Marin Cilic 6-4 7-5.
"I'm getting wins against quality players again and I had some doubts because when you've been through as many surgeries as I have and tough times, it makes it all the more sweet," said the former Wimbledon champion.
"Obviously it's going to be tough, he's one of the favourites to get a medal here," he said of his next opponent.
"But I'm going to go out and play my game. I've got nothing to lose."
Belarusian Azarenka stormed to a 6-1 6-2 victory over Spain's Maria Jose Martinez Sanchez, while third seed Maria Sharapova was taken to a first set tiebreak by world number 96 Brit Laura Robson before securing a 7-6 6-3 win.
American Williams also marched through her second round match against Canada's Aleksandra Wozniak with a 6-1 6-3 win before stopping at the side of the court to swap Olympic Committee pins with two journalists from the Bahamas.
"You've got to take advantage while you can," she said, laughing.
Later she returned to court with Beijing doubles medal partner sister Serena, defeating Germany's Angelique Kerber and Sabine Lisicki 6-2 7-5 to book a place in the quarter-finals.