Ashleigh Barty rallied for a 6-3, 2-6, 6-2 upset on a humid afternoon that played to her advantage. Barty, ranked No. 48 on the ATP tour, had never beaten a Top 10 player — going 0-6 — before knocking off the ninth-ranked Williams.
Several more upsets further depleted the brackets Wednesday. Numerous top players have pulled out because of injury, leaving the tournament wide open.
No. 3 Angelique Kerber lost to Ekaterina Makarova 6-4, 1-6, 7-6 (11), with the Russian converting on her eighth match point to end a 2-hour, 39-minute match. Makarova also upset Kerber at Roland Garros this season.
Kerber fell behind 5-2 in the third set, rallied to a tiebreaker, then got up 8-7. She wasted her only chance to finish it off.
"The third set was a completely up-and-down set," Kerber said.
On the men's side, wild card Frances Tiafoe broke through against No. 4 Alexander Zverev, who was coming off his second ATP Masters title in Montreal. The 19-year-old American hadn't taken a set in his two career matches against Zverev, but rallied for a 4-6, 6-3, 6-4 victory that ended Zverev's streak of 10 straight match wins.
The men's bracket is without Roger Federer, Andy Murray, Novak Djokovic and defending champion Marin Cilic because of injuries. The women's bracket is missing Serena Williams, Victoria Azarenka and Maria Sharapova.
Venus Williams is enjoying a comeback season, reaching the finals at the Australian Open and Wimbledon at age 37. She faded in the heat and humidity on Wednesday against a player who is 3-13 career against Top 20 players.
"I'm going to take a break," Williams said. "Just have a rest and just go big. That's my plan."
After knocking off Williams, Barty had to head for her doubles match, still buoyed by the biggest win of her singles career.
"It really is a privilege to be able to share the court with Venus," Barty said. "She's an amazing champion and how she's still playing so well, still making Slam finals — it's amazing. To be able to share the court with her and play well today, it was really exciting."
Madison Keys reached the round of 16 for the first time, needing just 50 minutes to beat past Daria Kasatkina 6-2, 6-1. Keys, who withdrew from last week's Rogers Cup in Toronto with a forearm injury, next will meet fourth-seeded Garbine Muguruza, the Wimbledon champion.
The men's bracket lost one of its hottest player of late. Zverev won back-to-back titles in Washington and Montreal, leaving him little time to rest. It showed against Tiafoe, who was more aggressive as the match went along.
"I'm completely dead right now," Zverev said. "I have been dead for the past two days. I could have easily pulled the plug in the second set, but I'm not going to retire just because I'm tired. And he's too good of a player to beat when I'm in this stage."