"I was prepared that I'm going to play Venus. It's going to be tough, because all the people are cheering for her," said Pliskova.
Pliskova, 24, who reached last eight in major for first time in her career as she had never been past the third round in 17 appearances at majors before.
Williams, 36, failed to convert a match point as she led 5-4 at the second set while Pliskova was serving at 30-40. But Pliskova erased that with a swinging forehand volley and followed it up with another forehand winner.
"I really played the perfect point there, and she managed to stay alive," Williams said about the match point she missed after the game.