In the super tiebreaker, the Czechs won four straight points to lead 9-5 and earned the title when Mladenovic netted a backhand volley.
It was the first Grand Slam title for Cermak and the second for Hradecka, who also won the women's doubles title at Roland Garros with Andrea Hlavackova in 2011. The pair lost in the Australian Open final in January.
After the match, Cermak and Hradecka kissed the Coupe Marcel Bernard. Their partnership has improved since their first match together five years ago, when they lost in the first round at Wimbledon.
"We didn't start well," Cermak said, with a laugh. "We're improving."
Hradecka still has a shot at the women's doubles title. She and Hlavackova will play their semifinal on Friday.
Nestor and Mladenovic led 4-0 in the first set. Cermak dropped serve in the opening game by netting three volleys and Hradecka double-faulted on break point. They took the first set when Cermak sent a forehand volley wide.
However, the Czechs broke Nestor in the opening game of the second. While serving for the second set, they rallied from 0-40 to even the match on a backhand volley from Hradecka.
"I'm a little bit upset that I lost serve first game in the second because that kind of changed momentum, although we did have some chances to break back," Nestor said.
Nestor won the men's doubles title four times at Roland Garros. He teamed up for the first time with Mladenovic, who at 20 is half his age. Despite the loss, the Canadian veteran pledged in the post-match news conference to play mixed doubles with Mladenovic at Wimbledon.