All four of their previous meetings went to three sets and this one was no different, though Williams looked the better of the two early - she squeaked out the first break of the match to take the first set, 7-5, and was just points away from a straight set win as the second set headed to a tie-break.
But Kvitova won that tie-break and, after the two kept holding serve for the first 11 games of the third, the No.6-seeded Czech finally broke to finish off the No.30-seeded American, 5-7, 7-6(2), 7-5.
"I knew she would play a great match on the grass today," Kvitova said. "She definitely loves it. Same as me. And I think the match showed just how great of a battle it was from both of us today."
"I think she played well at every single moment. There weren't a lot of opportunities for either one of us," Williams said. "I did the best I could, but she played really well. It just wasn't enough.
"But this is a great match for me. I'm looking forward to trying to play a lot of tournaments in the summer so I can just improve. I've hardly played any matches, so I think I'll just keep improving."
Kvitova was asked about Williams, who continues to try and fight her way back to the top.
"I think she's trying to come back. And she's doing a good job, actually," Kvitova said. "She's playing really, really well. She showed it as well today. I think that she can win some titles again.
"If she stays healthy and plays matches and everything like that, she can do it."
There were mixed results for the other top seeds in action, with No.4 seed Agnieszka Radwanska rolling past Portuguese qualifier Michelle Larcher de Brito in 57 minutes, 6-2, 6-0, but No.2 seed Li Na losing to Barbora Zahlavova Strycova, 7-6(5), 7-6(5), her second straight early exit at a major.
There was some last-minute drama in the match, Zahlavova Strycova thinking she had the match but then the call being challenged, changed, and the match resuming - Li then double faulted to end it.
"I think I did great today mentally," Zahlavova Strycova said afterwards. "It wasn't easy. The last point she challenged the match point and I thought I won the match, and I didn't hear that she challenged it, and I looked at the referee, and then it was in. It was really tough - you kind of won the match, but then you didn't win it. And it was so far away still - I was thinking to myself, like, 'Oh my gosh.'
"But I wasn't nervous at that point. I knew what I would have done."
It was Zahlavova Strycova's first win over a Top 10 player in 25 tries.
The other matches went mostly to form, with No.16 seed Caroline Wozniacki beating Croatian qualifier Ana Konjuh, 6-3, 6-0, No.22 seed Ekaterina Makarova beating Caroline Garcia, 7-5, 6-3, Peng Shuai beating Lauren Davis, 0-6, 6-3, 6-3, and Czech qualifier Tereza Smitkova beating Bojana Jovanovski, 4-6, 7-6(5), 10-8. The 19-year-old Smitkova is playing the first Grand Slam main draw of her career.
The last two second round matches were also played earlier in the day, with No.3 seed Simona Halep grinding past Ukrainian qualifier Lesia Tsurenko, 6-3, 4-6, 6-4, and Belinda Bencic beating American qualifier Victoria Duval in a close battle between WTA Rising Stars out on Court 18, 6-4, 7-5.