A tense clash between the two former grand slam champions saw Kvitova save two match points to force a deciding tie-break before eventually taking her fourth match point in a 5-7 6-3 7-6 (10) victory.
Kvitova watched Williams’ emotional final match against Ajla Tomljanovic on Friday evening, when she battled to the end, finding winners on five match points.
“I saw those match points when she didn’t give up,” said the Czech, who was in tears at the end. “She always hit a beautiful return. It was very special, she didn’t want to leave the court, for sure.
“That was the same case with me today. I didn’t want to go out of this tournament, so I was just there hanging somehow. She was playing aggressively, as well. Me too.
“So I was trying to go for it with these first serves or for aces. I think it was nice to see her yesterday fighting until the end.”
It was tough loss for Muguruza, who ended last season by winning the WTA Finals but has struggled all year.
She took encouragement, though, saying: “I have had some tough losses this year but definitely this one makes me feel like my fighting spirit was there, my tennis was there. I probably need some extra confidence playing on those match points.”
Kvitova next meets American eighth seed Jessica Pegula, who has become one the most reliable WTA players, while three-time finalist Victoria Azarenka is showing good form again in New York and eased to a 6-3 6-0 victory over Petra Martic.
Last year’s US Open was all about the rise of the teenagers in Emma Raducanu and Leylah Fernandez, and Kvitova is pleased to see the 30-somethings holding their own this time.
“I’m very happy for me, for Vika (Azarenka), and this kind of age,” said the Czech. “I feel like it’s good because we are on the tour so many years, and I’m just happy that we could still manage it and face the young generation.”
World number one Iga Swiatek was given her first test of the tournament by American Lauren Davis but won the final five games, fighting back from 4-1 down in the second set to triumph 6-3 6-4.
She next faces young German Jule Niemeier, who has backed up her run to the quarter-finals of Wimbledon by making the last 16 on her main-draw debut here, beating Zheng Qinwen 6-4 7-6 (5).
Sixth seed Aryna Sabalenka, who had to fight back from 6-2 5-1 down against Kaia Kanepi in the previous round, had a much easier time of it against France’s Clara Burel, racing to a 6-0 6-2 win.