Just over 24 hours after entering administration for a second time in two years, Pompey battled bravely with 10 men following Dave Kitson's red card in the first half before goals in the last 15 minutes from Jim O'Brien and Matt Done consigned them to defeat.
Portsmouth administrator Trevor Birch watched on from the main stand at Oakwell as around 400 travelling supporters chanted 'we will never die', but the immediate future looks bleak for the 2008 FA Cup winners, who are now in the bottom three.
Pompey boss Michael Appleton could only name three players on the bench, including goalkeeper Jamie Ashdown, and Kitson's soft red card for two bookable offence left them with a mountain to climb at Oakwell.
Portsmouth are now without a win in four matches, while victory for Barnsley got them back on track after successive defeats.
Craig Davies came agonisingly close to giving the Tykes a fifth-minute lead when he controlled the ball on his chest and beat Pompey goalkeeper Stephen Henderson with a dipping 20-yard shot that rattled the crossbar.
But that was a rare moment of quality in a dire first half, with Pompey's sole opportunity coming after 18 minutes when Joel Ward raced away down the right wing and his cross fell nicely for Erik Huseklepp, but the Norway forward fired wide at the back post from just inside the penalty area.
But the biggest talking point of the first half came three minutes before the break, when Kitson went in late on midfielder Corey Smith and referee Eddie Idlerton wasted no time in showing the former Reading striker a second yellow card.
Barnsley were still lacking ideas going forward but they were almost gifted the lead 53 minutes in, when Ricardo Rocha's header was heading into his own net before Henderson intervened by tipping the ball around the post.
But Pompey were far from being overrun and the lively Ward almost caught out Luke Steele with a vicious right-foot drive that the Tykes keeper palmed away for a corner.
Appleton's side looked destined to secure at least a point but Barnsley finally broke the deadlock with a goal from out of the blue 15 minutes from time as O'Brien let fly with his right foot from 20 yards out and found the bottom-left corner.
And Done sealed the points in stoppage-time as Pompey pressed forward and were caught on the break as the Barnsley substitute raced clear and sealed the points with a neat left-foot finish.