Williams was beaten 6-2, 7-5 by United States compatriot Sofia Kenin, who wasn't even born when the American legend made her Paris debut in 1998, in the third round.
Tennis star Naomi Osaka, who beat Williams and picked up her first major title at the 2018 U.S. Open, was also defeated in the third round of the French Open, losing to Czech player Katerina Siniakova.
The 37-year-old had not played two full matches in any tournament since the Australian Open in January and Kenin was simply far sharper and quicker around the court. Eleven of the top 16 seeds are also out, although last year's finalists-the champion Simona Halep and the runner-up Sloane Stephens-remain in contention. She lost 6-4, 6-2 in 77 minutes.
Being told of the decision prompted a furious reaction from Thiem, who said: "I don't really get it, seriously".
But needing time on court, she said she was thinking of playing a warm-up tournament this time around.
She went 3-1 ahead in the second set before Williams, the 2002, 2013 and 2015 champion, clawed her way back to 3-3.
There were no such problems for men's world number one Novak Djokovic who eased into the last 16, stepping up his bid to hold all four Grand Slam titles simultaneously for the second time.
West Indies tear through Pakistan, injury scare for Russell
After the fall of the first wicket, Babar Azam came to bat at number three and tried to move the scoreboard with Zaman. Our style is to be aggressive, whoever we are playing against. "If you contain any teams, you have to take wickets".
Williams was beaten 6-2, 7-5 by United States compatriot Sofia Kenin, who wasn't even born when the American legend made her Paris debut in 1998, in the third round. Russia's Vitalia Diatchenko in action during her first round match against Serena Williams of the U.S.
When Osaka remarked before the tournament that she sought a calendar year grand slam - last achieved by Steffi Graf in 1988 - it could have been interpreted as a tongue in cheek comment or at least a goal for further down the road.
Well, they said for now two to three weeks off, which means no tennis, of course, because it's my left forearm. Three points slipped away before she prevailed 6-3, 7-6 (4).
"I just think it was an unfortunate situation that I thought was amusing and we joked about it just before", Federer said. She took out Romanian Irina-Camelia Begu 7-6 (6), 6-4.
Williams, meanwhile, opened slowly in Paris and had to scramble from a set down to win her first match against Vitalia Diatchenko, then dispatched Japanese qualifier Kurumi Nara in more routine fashion - although nothing is coming particularly easy for her this season.
Wawrinka, the 2015 champion and a three-time major victor, recorded his 500th match win with a 7-6 (7-5), 7-6 (7-4), 7-6 (10-8) win over Grigor Dimitrov. "So everything definitely feels a little bit harder than normal, but at the same time, I know that it's going to get better".