Two second half goals from Benfica substitute Daniel Anjos sent Rovers crashing out of the Premier League International Cup.
Jacob Davenport slammed home midway through the second half to deservedly give the hosts a slender advantage in the Leyland drizzle.
Rovers were the better side throughout and had plenty of the ball, but were hit by a suckerpunch in the final 10 minutes when Anjos rose highest to head a cross beyond Andy Fisher.
The Brazilian frontman ended up having an ever greater impact later on when he fired home the dramatic late winner in added time with his right foot.
The result means that the final group game against Newcastle United next month will count for nothing as far as Rovers are concerned.
The positive of the night will have been a first run out in over a year for Jack Evans, whilst there was also a start for Andy Fisher in goal.
Jack Vale led the line, with support offered in the form of Dan Butterworth and Stefan Mols in the attacking areas.
A fine start from the hosts saw Davenport test Fabio Duerte with a curling free kick that the goalkeeper had to tip away for a corner after only 12 minutes.
Kevin Csoboth tested the returning Fisher at the other end before Vale forced Duarte into a low stop a moment later in a lively opening at Leyland.
Butterworth went close to opening the scoring just after the half hour mark, but dragged his effort just an inch or two wide of Duarte's right-hand post.
Chances were few and far between at the beginning of the second half, but it was Rovers who bagged the elusive opener through an expert finish from Davenport.
The midfielder won the ball back from an opponent before squeezing an effort under Duarte and into the back of the net just before the hour mark.
Skipper Mols could have made the score more comfortable for Rovers, but his strike belted against the base of the post before bouncing away from danger with less than 25 minutes to play.
Anjos had only been on for four minutes, but that was enough time for the big forward to power home a header with eight minutes remaining to level the score after a pinpoint cross from the left flank.
And the Brazilian wasn't finished there, with his impact ultimately winning the game for the Portuguese outfit as he beat Fisher with a right-footed attempt in the dying embers of added time.
Rovers Under-23s: Fisher, Rankin-Costello, Thompson, Davenport, Whitehall, Carter, Mols (c), Evans (Brennan, 90), Vale, Butterworth, Paton.
Unused substitutes: Hilton, Jackson, Pike, Boyomo, Saadi, Zimba.
Goal: Davenport, 57
Booked: Rankin-Costello, 57
Benfica Under-23s: Duarte, Ebuehi (J. Ferreira, 78), Ganchas, Conceicao, Frimpong (Anjos, 78), Pinto, Csobeth, Kalaica, Santos, Mendes (Vukotic, 70), Almeida.
Unused substitutes: Garrido, Chrien, V. Ferreira, Zec.
Goal: Anjos, 82, 90.
Booked: Frimpong, 71, Pinto, 90.