Ospreys scored a converted try in the 12th minute of added time to defeat Leicester 27-26 and book a place in the Champions League round of 16 on Friday.
Reduced to 14 men after a yellow card for replacement prop Dan Richardson, two-time European champions Leicester were unable to contain the rise of the Welsh team with full-back Jac Morgan scoring from close range.
Owen Williams then held back his nerve to kick the game-winning conversion.
“When the draw was made, most people would have thought that the later rounds in April and May wouldn’t worry us,” said Ospreys coach Toby Booth, whose team finished the group stage with three wins from four games. .
Two of those wins came against French champion Montpellier.
Leicester had already secured qualification for the knockout rounds before seeing their 100% winning record end.
“We are desperately disappointed as the defeat was our own fault as we opened the door for them and they are a good team in great form,” Leicester manager Richard Wigglesworth said.
“But we are in the knockout stages and we would have taken that result before the competition started.”
Jack van Poortvliet and Harry Simmons scored Leicester’s tries, and Springbok star Handre Pollard converted both and added four penalties.
Dewi Lake, Keelan Giles and Morgan crossed for the Ospreys. Williams kicked two penalties and two conversions with Cai Evans adding a conversion.
After three defeats to start the competition, French Lyon kept their slim hopes of reaching the round of 16 alive by defeating the South African Bulls 31-7.
Hooker Guillaume Marchand, with two tries, and scrum-half Jean-Marc Doussain scored in the first half for Lyon and a 24-0 lead at the interval.
Kurt-Lee Arend cut the Bulls’ arrears with his lone try before fly-half Leo Berdeu scored Lyon’s fourth just before the hour to secure an extra point.
The Bulls have yet to secure their place in the round of 16, while Lyon look set to fall in the knockout rounds of the Challenge Cup they won last season.
“Few people saw us qualify with a win against the Bulls,” Berdeu said.
“It doesn’t matter if we go back to the Challenge Cup. If we get down to that level, we’ll be looking forward to defending the title we won last year.”