For the second time in three games, Everton Under-23s were beaten by a penalty-kick deep into stoppage time.
The Blues led through Ellis Simms' strike on the hour but were pegged back 14 minutes later by Anthony Elanga's goal.
Everton defended resolutely at Leigh Sports Village but Ethan Galbraith broke their resistance from the spot in the 96th minute.
“To lose the game so late after the lads put so much effort into it was really disappointing and the boys didn’t deserve that,” said assistant manager John Ebbrell.
“It’s a difficult pill to swallow.
“United had good possession and moved the ball well but [goalkeeper] Joao Virginia has had very little to do. We protected him really well and I thought we always had a threat on the break.
“All we’ve done afterwards is try to pick the lads up in the dressing room because when young players put a maximum amount of effort in like that, there’s not much you can say.”
The contest was a slow burner with neither side creating anything of note in the opening quarter.
Top scorer Simms hooked an early effort wide and Einar Iversen was pressurised into rushing his shot from 10 yards and the ball sailed over.
On 36 minutes Beni Baningime played the ball to Tyler Onyango and his perfectly weighted pass set Simms through on goal, only for Ondrej Mastny to dash off his line and block the effort.
Argentinian international Marcos Rojo came closest for United in the first 45 minutes when his direct free-kick flashed inches past Virginia’s left-hand post.
Nathan Broadhead had half a chance just 15 seconds into the second period when Everton swept forward from the restart, but his shot cleared the target.
Virginia was called on in the 52nd minute when he adjusted himself well to smother a shot from Galbraith that was deflected.
The opening goal came after an hour.
Broadhead’s effort was blocked and as the ball looped into the box, Simms headed it goalwards.
Mastny saved the attempt but the Blues' leading scorer squeezed home the rebound.
Simms should have made it two shortly after. Iversen played a lovely through pass and when United keeper Mastny rushed out of his goal to clear the ball he collided with a defender.
Simms had a free shot at goal, but he over elaborated and allowed Brandon Williams the chance to clear off the line.
Sadly, Mastny came off badly in the collision and was taken off on a stretcher.
Elanga scored the equaliser in the 74th minute, finding space on the left of the Everton box before smashing a fierce shot past Virginia.
Substitute Mark Helm almost nicked it for United in the 90th minute but his well-struck shot was superbly turned away by a full-stretch Virginia before Everton were hit by United's late sucker punch.
Williams dribbled the ball into the box and went down under a challenge from Imam Jagne. Virginia guessed right from Galbraith’s kick but the ball found the net for the cruellest of finishes.
“I’m not so sure it was a penalty, it was a 50-50 one, but the referee has given it,” said Ebbrell.
“The lad has ‘won’ it and you can’t knock him for that because he did well in his situation and used his experience.
"We’re all disappointed but there’s an organisation and a real spirit amongst the lads.
"They’ve put absolutely everything in tonight and we know they’ll come back as soon as we start training and preparing for the next game.”