Substitute Jason Scotland grabbed a 74th-minute winner for the relegation-threatened Tykes with his third goal in as many games to complete a pulsating second half at the Riverside Stadium.
Middlesbrough have now lost all five of their league games in 2013, and were punished for failing to hold onto their lead after overturning a 1-0 first-half deficit.
Jim O’Brien’s deflected effort gave Barnsley a fifth-minute lead, and the home side looked bereft of ideas in the opening period.
They improved after the break and fought back through Mustapha Carayol and Curtis Main.
However, having gone in front Boro were punished for resting on their laurels as Scott Golbourne bundled home after Jason Steele failed to deal with Rory Delap’s long throw, before Scotland sealed the hosts’ fate from an indirect free-kick after the Boro keeper picked up Rhys Williams’ back-pass on the edge of his six-yard box.
Only goal difference keeps Barnsley in the relegation places, but Flitcroft was quick to warn his players against complacency.
“It’s got to be 46 games and this group of players will not get carried away,” he said. “I won’t let this group of players get away with anything; that’s my job as the manager.
“I don’t do people who come to work to be average - I do people who come to work to achieve, whoever that person is.
“The game had everything. Like any great rollercoaster it had a bit of everything in it.
“Every player played a massive part in that win - that’s a special group of players in there.
“This group of players are achieving their potential week in, week out, and that’s all I ask. If you give everything you’ve got you hit the levels of perfection that I talk about to these players.
“I’m telling this group to become heroes and achieve something that at one stage looked impossible and gets talked about for years to come.”
Flitcroft also praised former Barnsley boss Keith Hill, who was sacked in December with the side bottom of the table and four points adrift of safety.
“Keith Hill’s assembled an incredible squad - his recruitment is amongst the best in the country,” added Flitcroft. “Fortunately now we’re getting the best out of them.
“We’ve played well all season, and I’ll stress that to anyone who wants to listen.”
A despondent home crowd were quick to become aggrieved after O’Brien’s opener, as the home side’s efforts were undermined by a string of wayward passes and a chronic lack of creativity in the final third.
Carayol’s equaliser was Boro’s first Championship goal for 230 minutes.
However, once Main had bundled the home side ahead it was defensive problems, rather than their shortcomings going forward, which cost Boro, and Mowbray was left bemoaning their luck after O’Brien and Scotland’s goals took deflections.
“Managers at the bottom end of the table normally come in and complain about things going against them - deflected goals and a bit of bad luck - I feel a bit like that at the moment,” said Mowbray, whose side remain in the play-off places despite another defeat.
“It was a kick in the teeth after five minutes with the huge deflected goal.
“We’ve found it hard work first half, a frustrating 45 minutes, but did well to get back in front so to lose it from that point is very, very disappointing.
“I can’t question their commitment to win the game; I can feel their frustration in the dressing room at the end. We’ve given everything we can to try and change it around.
“How do you get out of it? You stick together, you work hard, and you try to do the right things every day. You have to keep going.”