The 24-year-old left-hander, who a year ago needed wrist surgery, on Saturday proved too steady for the error-strewn Jabeur, who ended up as runner-up as she did in 2022.
Jabeur, beaten by Elena Rybakina last year and by Iga Swiatek in the 2022 US Open final, was bidding to become the first Arab player to win a Grand Slam title and the first African woman to lift one of the four major trophies.
But Jabeur, 28, was well below her best with 31 unforced errors killing her chances of a victory that would have been a milestone moment for women's sport.
"This is the most painful loss of my career," the crowd favourite said as she fought back tears.
"Today is going to be a tough day for me but I'm not going to give up and I am going to come back stronger. It's been a tough journey but I promise I will come back and one day win this tournament."
The unassuming Vondrousova had managed to win only one match in her previous four visits to Wimbledon and last year came with her wrist in a plaster cast to support a friend and go shopping.
But she beat four seeds during the fortnight, including number four Jessica Pegula in the quarter-finals and Ukraine's crowd darling Elina Svitolina in the semis.
"I don't know what's happening right now," Vondrousova, whose husband Stepan Simek was present for the final after spending the past two weeks looking after their pet cat Frankie back in Prague, said on court after receiving the Venus Rosewater Dish from Catherine, Princess of Wales.
"This time last year I had a cast on so it's amazing that I can now stand here and hold this, it's crazy. I don't know how I've done it."
Rain threat
With the Centre Court roof shut because of high winds and the threat of rain showers, overwhelming crowd-favourite Jabeur initially looked comfortable as she won the opening two games of the showpiece.
But world number 42 Vondrousova, playing a canny game full of slow slices and clever angles, settled down and began to draw errors from her opponent.
Jabeur responded again to lead 4-2 but then seemed consumed by nerves as mistakes flowed from her racket and she dropped five successive games to lose the opening set
Vondrousova, sporting tattoos on her arms one of which says "No Rain No Flowers", led 1-0 and 40-0 in the second set and appeared to be marching to victory as Jabeur fell apart.
But suddenly the wily Tunisian loosened her shoulders and found her range to seize back control.
It looked as though Jabeur was getting on top as she won three games in a row, but Vondrousova never panicked.
Showing great court craft and subtle changes of pace she got back to 3-3 and as Jabeur's errors returned with a vengeance she broke serve at 4-4 to stand one game away from the title.
Reaching 40-0, she squandered her first match point with a double fault but put away a volley to claim the title at the second time of asking, appearing in disbelief.
Vondrousova, the first player to reach two Grand Slam finals as a non seed having contested the 2019 French Open decider, became the fourth Czech-born player to win the title in the professional era after Martina Navratilova, Jana Novotna and Petra Kvitova.
She was also the sixth successive first-time winner of the women's crown.
Beer and a tattoo
Asked on court how she would celebrate her victory, a smiling Vondrousova said: "I think I'm going to have some beer maybe. It was an exhausting two weeks, and I was getting myself together the last few days. I was so nervous before this game."
Vondrousova has a collection of tattoos on her arms and said she was thinking of getting another one to celebrate a memorable fortnight in London. Her coach Jan Hernych would be getting inked too after a pre-match pledge.
"I don't know but I made a bet with my coach. He said if I win a Grand Slam he's going to get one also. So I think we're going to go tomorrow."
After knocking out four seeds en route to the final and then out-playing Jabeur to become the fourth Czech-born woman to win the title in the professional era, the celebrations will be well-deserved. And the timing could hardly have been better.
"It's amazing, tomorrow is our first anniversary of our wedding. It's amazing to have you guys being here with me," she said looking up to Simek and family members in her box, although her mother Jindriska had preferred to watch on television at home.
Later, after waving to crowds from the Centre Court balcony, she told reporters she had been surprised to see her husband shed a tear, especially as he had been remarkably calm throughout the contest.
"I think when I came to the box, he cried. I saw him after, and he cried a lot. I think that's the first emotion I saw (in) him over the eight years," she told reporters.
"I think he cried on the wedding day also but that was it."
With a cheque for £2.3 million ($NZ4.7 million) in the bank, Vondrousova said she would buy some "nice fish" for her pet cat. "I think she doesn't really care what's happening, but, yeah, I'm going to buy her something good."
