Andrea Pratichetti crossed late as Treviso fought back to snatch a shock victory 17-14 over the Ospreys.
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The Welsh region appeared on course to finish their Heineken Cup campaign on a high thanks to Tom Isaacs' try and the boot of Dan Biggar.
But Treviso launched a late comeback with 14 unanswered points in the final five minutes of a previously uneventful clash at a muddy Stadio Comunale di Monigo.
First Alessandro Zanni crossed the whitewash following a sustained period of pressure and the Italians completed their miraculous recovery with Pratichetti's intervention with just five seconds to spare.
Ospreys had no chance of qualifying for the knockout stages of the Heineken or Amlin Challenge Cup but were determined to finish a promising European campaign on a high.
Biggar and new Wales squad member James King started for the Ospreys, but they rested Justin Tipuric and star trio Ryan Jones, Ian Evans and Richard Hibbard were all ruled out through injury.
They were dealt a further blow in the hours before kick-off with wing Eli Walker forced out due to a hamstring strain.
Treviso put the first points on the board with a Kris Burton penalty after four minutes, but Biggar, in line to pull on the number 10 jersey for Wales in the RBS 6 Nations, slotted two quick penalties in reply to give Ospreys the lead.
Both sides threatened in a frantic close to the half but the score remained unchanged.
The Ospreys almost surged further in front with Kahn Fotuali'i powering through. The scrum-half, who is being courted by a number of top European clubs, bundled through several defenders from the back of a rolling maul but was held up short.
Steve Tandy's men continued to pile on the pressure following the ensuing scrum and they eventually settled for a Biggar drop-goal.
They took a major stride towards victory on 58 minutes as Isaacs raced clear. Ben John burst through Ludovico Nitoglia and offloaded inside to Fotuali'i, who demonstrated quick hands to release Isaacs for the score.
Treviso, who flexed their muscles with several rolling mauls throughout the clash, rumbled over on 70 minutes but replays were inconclusive and referee Greg Garner was unable to award the try.
But they eventually manufactured a breakthrough as Zanni powered over to set up a tense finale.
And with their tails up, Treviso rampaged up field with Giulio Toniolatti and Valerio Bernabo both prominent in the build-up to Pratichetti's match-winning score.