Roddick, a three-time champion of this U.S. Open Series event, came out flat against the 13th-seeded Frenchman in a match that started just before 11 p.m. because of a 4½-hour weather delay.
Roddick, ranked ninth in the world, was the third highly seeded American to lose over the past two days.
"It was just a bad night," Roddick said. "I don't really have any defense for it. I didn't feel right physically. I didn't feel right mentally. I wish I had answers for you right now, but I just don't.
"[But] I promise you, I'm going to figure this out before I do that again."
Roddick, who was 26-4 through April, has lost five of his past 14 matches on tour. He had won his previous two matches against Simon in straight sets.
"The difference is that I was able to return his serve today," Simon joked about the match that ended just before 1 a.m. "He wasn't able to hit winners versus me because the court was a little slow and I was moving well."
In other matches Thursday, top-seeded Tomas Berdych of the Czech Republic beat 16th-seeded Andrey Golubev of Kazakhstan, 6-3, 5-7, 7-5; and third-seeded Fernando Verdasco of Spain defeated American Ryan Sweeting 6-4, 7-5.
Malisse overcame 18 aces by Isner and took advantage of a number of mistakes by his 6-foot-9 opponent, who had won each of his seven previous tiebreaks at this event.
"He serves well, and because of that you have a little more pressure on your serve," Malisse said of Isner, who leads the ATP tour in aces. "I just tried to get the serve back. That's his big weapon. You take what you can get, and right now I'm just happy to be in another quarterfinal."
The weather delay might have affected the players.
"I don't know, maybe," Verdasco said. "Of course waiting is not an easy thing."
The three doubles matches scheduled for Thursday were pushed to Friday.