The American star saw her latest attempt to win the title at Roland Garros come to an end in the fourth round on Sunday when she lost to 30th-seeded Timea Bacsinszky of Switzerland 5-7, 6-2, 6-1 on Court Philippe Chatrier. It was the second year in a row Bacsinszky beat the No. 10 seed Williams in the fourth round in Paris.
Williams, who turns 37 this month, is a seven-time Grand Slam champion -- all of those titles came at Wimbledon or the U.S. Open. Her best French Open showing was in 2002 when she lost to her sister Serena in the final.
Also, Kristina Mladenovic thrilled the home crowd with the biggest win of her career, knocking off defending champion Garbine Muguruza 6-1, 3-6, 6-3 to advance to the quarterfinals.
The 13th-seeded Mladenovic will next face Bacsinszky, whose best result at a major tournament was reaching the semifinals at Roland Garros in 2015.
Meanwhile, Williams won the first set against Bacsinszky before being outplayed in the final two of the two-hour, 12-minute match.
Bacsinszky opened up a 4-0 lead in the third set before wrapping up the match. Williams committed 45 unforced errors in the defeat.
"She just played well," Williams said afterward. "I think there were just some points where she just came up with great tennis a lot of the times in the second and the third set. You know, sometimes it's just bad luck."
The ousters of Williams and Muguruza mean that none of this year's eight quarterfinalists have ever won a Grand Slam title. The last time that situation occurred was at the 1979 Australian Open when Barbara Jordan won her first Grand Slam crown.
Muguruza, the fourth seed from Spain, helped create that situation as she ran into a determined Mladenovic with the Frenchwoman backed by a vocal crowd of countrymen in Paris. The 24-year-old Mladenovic reached the quarterfinals at Roland Garros for the first time.
Mladenovic pulled off the victory despite 16 double-faults, seven in each of the last two sets at Court Suzanne-Lenglen.
Mladenovic broke down in tears during her post-match speech to the crowd as they yelled her nickname, "Kiki, Kiki."
"I love you, too," Mladenovic told the chanting crowd after the win. "You make me cry.
"Let me try to speak. It's a last-16 match against the defending champion. Big match, which I was waiting for. It's not perfect, there are little problems but I fight as I can. ... You give me so much strength."
Muguruza beat Serena Williams in the French Open final last year. The last repeat women's champion at Roland Garros remains Justine Henin, who won the tournament three consecutive years from 2005 to 2007.
"I couldn't really find my game, but I don't think I did really something wrong out there," an emotional Muguruza said after the match. "You know, I just think it went to her side, and that's all. There was a few points that she played well at the end of the match, and kind of that was it."
The 11th-seeded Wozniacki had 15 fewer unforced errors than No. 8 Kuznetsova.
After her win, Wozniacki was asked about Saturday night's London terrorist attack that left seven people dead and dozens more injured.
"It's obviously very awful what's happening or what's happened the last few weeks and obviously what happened here (in Paris) in the past, as well," Wozniacki said. "You know, I don't know what you can do to prevent it.
"At the end of the day, I think they are here and the UK trying to do everything they can to keep the security at the highest level, and I think here they are doing a good job. I'm sure that when we go to the UK to play, they will do a great job, too."
Wozniacki will next face Latvia teenager Jelena Ostapenko, who beat No. 23 seed Samantha Stosur 2-6, 6-2, 6-4 of Australia. The 47th-ranked Ostapenko is the youngest player in the tournament at age 19.
Second-seeded Karolina Pliskova of the Czech Republic is in a good position after stopping Germany's Carina Witthoeft 7-5, 6-1.
Last year's U.S. Open finalist is the highest seed remaining and she looked strong as she won 11 of the final 13 games against Witthoeft.
"I am surprised that I'm in the fourth round here," Pliskova said after her match. "For the first time, obviously. I'm also quite surprised that, at three straight Grand Slams, I passed the third round. But especially on clay, I've never passed the second here. So it's quite huge for me."
Pliskova doesn't want to hear chatter about now being the favorite as she openly admits clay isn't her favorite surface.
"I don't really feel I am favorite on paper on the clay," Pliskova said. "So every match for me is tough and it doesn't matter if she's (ranked) 60 or if she's 300.
"I still don't think I'm playing the best tennis. It's good, but I can still improve. So definitely today, the second set I think was the best so far that I played in this tournament. But still there are some things that I want to improve and that I want to play better."
Pliskova will next face Paraguay's Veronica Cepede Royg, who beat Mariana Duque-Marino of Colombia 3-6, 7-6 (2), 6-3.
Fifth-seeded Ukrainian Elina Svitolina continued her hot streak by defeating Magda Linette of Poland 6-4, 7-5.
It was the eighth consecutive victory for Svitolina, who was won four tournaments this year.
"It was very strange match, because she was playing good and I was playing OK, but I think today was enough," Svitolina said. "For me, on important moments, I played very good and that's what made the difference. But for the rest, you know, my game was I showed today was enough, and I was very happy that I could finish in two sets."
Svitolina next faces Croatian qualifier Petra Martic. The 26-year-old Martic upset 17th-seeded Anastasija Sevastova of Latvia 6-1, 6-1 in one of four women's third-round singles matches washed out by rain on Saturday.