The Czech has become the first defending women's champion to be beaten in the opening round this century, and only the second since the Open era began in 1968.
Vondrousova, who was seeded sixth this year, was beaten 6-4 6-2 by Jessica Bouzas Maneiro.
Jessica Bouzas Maneiro can barely believe she has beaten the Wimbledon champion Marketa Vondrousova. (AP PHOTO)
Vondrousova's predecessor as champion, Elena Rybakina, cruised past Romanian Elena-Gabriela Ruse 6-3 6-1.
There were no problems either for top seed Iga Swiatek, who began her quest for a maiden Wimbledon crown with a 6-3 6-4 victory over American former world No.4 Sofia Kenin.
World No.1 Iga Swiatek made a winning start to her fifth Wimbledon campaign, beating Sofia Kenin. (AP PHOTO)
While they look forward, a disconsolate Vondrousova looked back.
"It's tough to go out defending the title," she said. "I was really nervous from the morning and everything. She was also playing a good match. That was kind of tough, too.
"It's tough feelings also to go back, I feel like everybody just expects you to win maybe. I was happy to be back on the Centre Court. It just didn't go as planned."
The last champion beaten at the first hurdle was Steffi Graf, who lost to Lori McNeil in 1994.
Bouzas Maneiro is 83rd in the world, a career-high ranking.
"This is one of the most important moments of my life, in my career here on this court, this is amazing," she said.
"The atmosphere here, the tournament is the most beautiful tournament I ever played in my life."
The 21-year-old Spaniard, added: "I had no pressure, enjoy the moment, enjoy the tournament and just trying to be free-playing and I did it."
World No.1 Swiatek has yet to shine on grass, having never reached a final on the surface, but any hopes of slowly finding her feet were dashed after the draw pitted her against the player who toppled Coco Gauff in the 2023 opening round.
But after an uncertain start in which both players dropped serve, the Pole settled to secure her 20th consecutive win.
Ons Jabeur blasted her way into the second round with a 55-minute, 6-3 6-1 demolition of Japan's Moyuka Uchijima. The Tunisian 10th seed, runner-up for the last two years, next meets American Robin Montgomery.
Caroline Wozniacki claimed a 6-2 6-0 win over Alycia Parks in the former world No.1 's return to Wimbledon after four years.
There was also an easy win for fifth seed Jessica Pegula, who beat fellow American Ashlyn Krueger 6-2 6-0.
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