Former champion Andy Roddick and women's title holder Maria Sharapova are also in action but Federer could have the toughest match when he faces up to 6-ft-9 Isner.
"I saw he had a good serve," Federer said.
"He's got a good second serve, too. It's going to be interesting to see how I handle that because the trajectory of a tall guy is always different. It's going to be interesting to see how good he is from the baseline."
Isner is playing just his fifth tournament as a professional, having reached the final in Washington earlier this month and risen from outside the world's top 800 in June to his current ranking of 184.
"I'm going to go out there and just play my game," Isner said.
"Most importantly, have a lot of fun. I'm going to believe. I'm not saying I'm going to win, but I'm going to believe. That's what I have to do."
Fifth seed Roddick faces former Australian Open champion Thomas Johansson of Sweden in another third-round match while fourth seed Nikolay Davydenko of Russia and American sixth seed James Blake also bid for places in the last 16.
Sharapova, who has impressed in her first two rounds, plays Agnieszka Radwanska, the Polish 30th seed, for a place in the last 16.
Former champion Martina Hingis, seventh seed Nadia Petrova and 13th seed Nicole Vaidisova are also in third-round action.
