That match saw Nadal, the French Open victor for the men, eliminated in five sets. She sealed the outcome of the game at the eighth attempt.
Their inspirational father and coach Richard has also had to miss this year's championships.
Ostapenko, 20, who reached this past May aged 19 and never having surpassed a Grand Slam third round in seven tries, now has surpassed two.
Wimbledon's second Monday traditionally features every fourth-round match in the men's and women's singles, which is a scenario that sets it apart from the other grand slams.
She responded by saying: "They were cheering for me". "It wasn't as easy maybe as it looks like".
No. 24 CoCo Vandeweghe was the second-biggest upset-maker after overpowering former No. 1 and No. 5 Caroline Wonziacki 7-6 (6), 6-4. "I think I'm again on a good way to playing again a high level of tennis".
"I definitely feel that age is not a factor with her", Konta said about Williams. "I was happy about that". Unfortunately, his success may not last long as his next opponent is Marin Cilic. The seventh-seeded Croat beat Roberto Bautista Agut 6-2, 6-2, 6-2.
The surprising defeat extended Nadal's drought without a quarterfinal berth at the All England Club to six years.
Play has started on Centre Court at Wimbledon with the roof closed. And although the world number 13 saved one set point with a victor into empty court, Williams promptly held to wrap up the opener in 31 brisk minutes. He will next face Canada's Milos Raonic.More news: Senate bill is evil
Williams has made $36.5 million on the court during her career, and even more from endorsements. Her Wimbledon debut came a few months before Konjuh was born.
She has beaten Simona Halep three times in four meetings, though Halep got the last one on Rome's clay. Halep also advanced Monday, while Pliskova lost in the second round.
Williams beat Jelena Ostapenko in straight sets to move into the Wimbledon semi-finals - leaving her just two games away from Wimbledon glory. Williams has all the experience, but Ostapenko is on an incredible run of form and her game tends to suit the grass courts.
Instead of four men's matches and two women's matches, I would like to see, and I think all women would like to see, three men's matches and three women's matches to go along with the equal prize money."
The second set provide a hint of the fearlessness which has brought Ostapenko among the world's best. If she wins Wimbledon, she'll make $2.8 million.
Next she will face the brightest of the sport's next-generation stars, French Open champion Jelena Ostapenko. However, she won the girls' title at the All England Club in 2014. "But I thought we would play on a bigger court". "Because of that I had more pressure because I had to keep my serve".
Otherwise, Djokovic had little trouble eliminating 51st-ranked Adrian Mannarino of France 6-2, 7-6 (5), 6-4 in a match postponed Monday night because of darkness.
Muguruza and Kuznetsova will meet in the next round. She will face the victor between American Coco Vandeweghe and Magadalena Rybarikova of Slovakia.