Mathieu topped Isner 6-7 (2-7), 6-4, 6-4, 3-6, 18-16. The match took 5 hours, 41 minutes to complete with the final set lasting nearly 2 1/2 hours. It was the most games in a French Open fifth set.
Mathieu had some success against Isner's serve and finished off four of his 24 break chances, including the final point of the match. He had a respectable 40 unforced errors in a 250-point match while Isner countered 98 unforced errors with 107 winners.
Isner managed just four break opportunities against Mathieu, getting one break each in the first and fourth sets. Mathieu won 76 percent of the points on serve.
No. 2-seeded and two-time defending champion Rafael Nadal roared through a 6-2, 6-2, 6-0 win in his match Thursday against Denis Istomin. Nadal took 56 percent of the points off Istomin's serve.
Jo-Wilfried Tsonga, the No. 5-seed, finished off a 6-2, 4-6, 6-2, 6-1 win over Cedrik-Marcel Stebe in a match that was suspended because of rain Wednesday while sixth-seeded David Ferrer handled Benoit Paire 6-3, 6-3, 6-2 and No. 8 Janko Tipsarevic defeated Jeremy Chardy 7-6 (7-4), 6-1, 7-5.
A series of upsets hit some of the lower seeds Thursday. Leonardo Mayer was a 7-6 (7-5), 7-6 (7-4), 7-5 winner over 24th-seeded Philipp Kohlschreiber; Santiago Giraldo knocked out No. 25-seed Bernard Tomic 6-4, 6-1, 6-3; Fabio Fognini took out 28th-seeded Viktor Troicki 6-2, 3-6, 4-6, 6-3, 8-6; and Eduardo Schwank upset 32nd-seeded Florian Mayer 6-2, 3-6, 6-4, 7-6 (7-4).
But 12th-seeded Nicolas Almagro, No. 13 Juan Monaco, No. 17-seeded Richard Gasquet, No. 19th Milos Raonic, 27th-seeded Mikhail Youzhny and No. 29 Julien Benneteau are all headed into the third round as are unseeded players David Goffin and Tommy Haas.