Karolina Muchova took advantage of an error-prone Venus Williams to advance 6-3 7-5 into the second round of the US Open on Tuesday.
With this, she handed the two-times champion her first opening round loss at the tournament.
Muchova broke Williams to start the match.
She deployed a wide variety of shot-making, including a pinpoint serve, a slice backhand and some well-timed trips to the net, to frustrate the former world number one.
Williams was sharper at the outset of the second set and served at 5-4 with the chance to extend the match to a third.
But a backhand sailed on break-point and Muchova leveled at 5-5.
The 24-year-old Czech held at love the next game and sealed the win when Williams issued her third double-fault on match-point.
“From the start I tried to be relaxed and get to my game, but it was tough to get into a rhythm because we were having very short rallies,” said Muchova, who hit 27 winners and saved six of her eight break-points.
“I was happy I was able to turn it on in the end.”
The 40-year-old Williams was 21-0 in opening round matches at the US Open coming into Tuesday.
But the seven-times Grand Slam champion was undone by 34 unforced errors at Arthur Ashe Stadium, where her sister Serena looked on from the largely empty stands.
Next up for the 20th-seeded Muchova is Russian Anna Kalinskaya, who defeated Nina Stojanovic of Serbia in straight sets earlier in the day.