Full-time score from The Den
Fred Onyedinma's first two goals for Millwall were not enough for The Lions as Lewin Nyatanga scored a cruel late winner to secure a 3-2 victory for Barnsley at The Den.
Onyedinma netted either side of goals from Sam Winnall and Alfie Mawson, but Nyatanga, in the second minute of added time, headed home at the far post to send Millwall home nursing back-to-back Den defeats in the League.
Neil Harris made three changes to the line-up which started Saturday's defeat to Coventry City, bringing in Sid Nelson, Shane Ferguson and the fit-again Jimmy Abdou for Byron Webster, Mark Beevers and Lee Martin.
The Lions launched an instant attack from kick-off which was led by Onyedinma, who beat two defenders down the right flank before eventually being dispossessed inside the 18-yard box.
Millwall kept up some early pressure on the away side's goal but Ed Upson and Steve Morison's attempt at a neat one-two on the edge of the area was well defended by The Tykes.
Lee Johnson's side made their first meaningful attack in the seventh minute when Marley Watkins charged down the right but his eventual cross into the box was well collected by David Forde.
The Irish keeper was again called into action two minutes later when he was forced to punch Daniel Crowley's cross clear before Nelson, in the same attacking move, made a vital tackle inside the area.
Suddenly in the ascendancy, Barnsley almost forced the opener from a goalmouth scramble but home fans were able to breathe a sigh of relief when Tony Craig arrived to thump the ball away.
At the other end, Onyedinma, Millwall's brightest attacking outlet in the early stages, sent a teasing cross into the area which just evaded the touch of Lee Gregory eight yards from goal.
Millwall began to increase their pressure on the Barnsley goal and they were rewarded for a spell of dominance 27 minutes in. Morison received the ball in the box, took his time and picked out Onyedinma, whose initial effort was blocked by Adam Davies before the winger managed to squeeze the ball over the line at the second attempt for his first senior goal for The Lions.
It was just moments later when Upson received the ball from Morison before producing a wonderful cross which Shaun Williams, at full stretch, could only turn narrowly wide.
The Lions had the ball in the net for a second time on 35 minutes but Craig's looping header, which was helped in by Onyedinma, was ruled out after the winger had strayed into an offside position.
But then, against the run of play, Barnsley were level. A floated cross found its way to the far post for Watkins, who took a touch before firing past Forde and into the bottom corner thanks to a slight deflection.
Neither side made any changes for the second-half and Barnsley had a chance to go in front just seconds in. Ben Pearson's long-range effort, though, ended up well wide of Forde's left-hand post.
Millwall's first chance of the second-half was so nearly a goal as Ferguson's whipped corner found Williams, whose bullet header was cleared off the line.
It was the visitors who went in front for the first time in the game on 57 minutes. The ball was worked out wide before finding its way to Mawson, who superbly volleyed past Forde and into the bottom corner.
With Barnsley growing in control of the game, Harris brought on Ben Thompson and Aiden O'Brien for Williams and Upson on the hour mark in an attempt to shift the momentum once again.
Thompson's first involvement was to track the run of Watkins, who charged into the area from deep before shooting just wide of the target.
At the other end, Gregory's dipping shot from distance, after bringing the ball down on his chest, edged narrowly wide from 25 yards as The Lions tried desperately to force a leveller.
And on 82 minutes, they did just that. Ferguson, a danger from set-pieces all night, swung in another corner from the right which was met by Onyedinma at the near post, who headed in his - and Millwall's - second of the night.
But in a cruel late blow Nyatanga headed a deep free-kick home at the far post to send the away fans into raptures.
: Forde, Cummings, Nelson, Craig, Ferguson (L.Martin, 89'), Onyedinma, Abdou, Williams (Thompson, 61'), Upson (O'Brien, 61'), Gregory, Morison. Subs not used: Archer, Webster, Cowan-Hall, Marquis
Scorers: Onyedinma 27' & 82'
Barnsley: Davies, Bree (Wabara, 58'), Roberts, Mawson, Nyatanga, Scowen, Pearson (Rothwell, 84'), Hourihane, Watkins, Winnall, Crowley. Subs not used: Townsend, Smith, R.Williams, Digby, Wilkinson
Scorers: Watkins 39', Mawson 57', Nyatanga 90+2'
Ref: D Whitehouse
Att: 7,657 (away 304)