The most significant result on the men's side was Grigor Dimitrov's 6-7 (1), 6-4, 6-3 victory over second-seeded Andy Murray.
Williams seemed to fade after a long first set that lasted nearly an hour.
Williams has failed to win any of the three tournaments she entered this year, and she has not won an event since capturing the tournament in Cincinnati in August.
"I did the best I could," Williams said in a brief postmatch news conference. "I can't win every match. These players come out and play me like they've never played before in their lives. I have to be 300 percent every day."
The 15th-seeded Kuznetsova finished with only 18 unforced errors compared to 55 for Williams.
Williams' serve was not the weapon it usually is. She hit 13 aces but also double-faulted nine times and had her serve broken six times.
"Physically I'm fine," she said. "I don't know, I guess I didn't move today. Maybe that was one of the things that didn't work out for me."
Kuznetsova improved to 3-8 against Williams and beat her for the first time since the 2009 French Open quarterfinals.
"I'm too old," the 30-year-old Kuznetsova told the crowd. "I've been many times on this court. I love being back here, and I'm really happy with my performance."
Third-seeded Agnieszka Radwanska was also eliminated Monday, falling to No. 19 Timea Bacsinszky 2-6, 6-4, 6-2.
Second-seeded Angelique Kerber needed three sets to hold off Timea Babos 6-2, 3-6, 6-4.
Perhaps the most intriguing match was 13th-seeded Victoria Azarenka's 7-6 (6), 7-6 (4) victory over fourth-seeded Garbine Muguruza. Azarenka is currently ranked No. 8 as she makes her way back up the rankings.
In other women's matches, No. 22-seeded Madison Keys of the United States defeated Irina-Camelia Begu 6-3, 6-1; No. 5-seeded Simona Halep beat Heather Watson 6-3, 6-4; Ekaterina Makarova ousted 12th-seeded Elina Svitolina 6-1, 6-4; and Johanna Konta eliminated Monica Niculescu 6-2, 6-2
On the men's side, the 26th-seeded Dimitrov provided the headline by beating Murray.
"To be honest, I just played better in the big moments today," Dimitrov said. "I had quite a few opportunities and I used them."
Murray led 3-1 in the third set but let it slip away.
"A lot of unforced errors in the third set," Murray said. "Obviously, I didn't start the second set particularly well (getting behind 4-1). After winning a close first set, you want to try and put your opponent under pressure.
"Credit to him. He was more solid than me."
In other men's matches, No. 6 seed Kei Nishikori beat No. 27 Alexandr Dolgopolov 6-2, 6-2; ninth-seeded Jo-Wilfried Tsonga was upset by 17th-seeded Roberto Bautista Agut 2-6, 6-3, 7-6 (4); fourth-seeded Stan Wawrinka topped unseeded Adrian Mannarino 2-6, 7-5, 6-0; 12th-seeded Milos Raonic defeated 22nd-seeded American Jack Sock 7-6 (3), 6-4; 24th-seeded Nick Kyrgios of Australia beat qualifier Tim Smyczek 6-4, 6-4; and Damir Dzumhur knocked off qualifier Mikhail Kukushkin 4-6, 6-1, 6-2.