The 23-time Grand Slam champion fought back from a set down before losing 3-6 6-2 3-6 to the Czech in an entertaining contest at the Western and Southern Open.
Kvitova picked up just her second win against the American great in seven attempts, although she has now picked up back-to-back victories over the 36-year-old.
Williams had responded well to the heaviest defeat of her career against British No. 1 Johanna Konta in San Jose by thumping Daria Gavrilova in the first round and she put in a decent performance against 2018 WTA title leader Kvitova.
And though she fell short, she was happy to perform at a ‘really high level’ and believes there’s a long, exciting road ahead of her.
‘I think it was a good match today,’ she said. ‘We both played at a really high level. Only one person could win.
‘You know, this is a long comeback. I just began. I just started — definitely at the very, very beginning.
‘I’m getting there, and I’m going to just continue to work hard and, hopefully, I’ll start winning more matches.’
Two-time Grand Slam winner Kvitova – who will go on to face Kristina Mladenovic in the third round – was full of praise for Williams, who forced her to dig deep to claim her 40th match win of the season.
‘It was a pleasure to share the court with Serena, she’s such a champion,’ Kvitova said.
‘We are both coming back – from injury, from motherhood – so it’s something really special to play her.
‘Unfortunately it’s in the second round, but playing a night session against Serena in front of a USA crowd, it’s been great.’