Barnsley lost 3-2 at Portsmouth, having led 2-1 on 85 minutes, and still need three points from the last two games to secure a play-off place.
Devante Cole ended his goal drought with a fifth-minute opener but the hosts levelled almost instantly. John McAtee restored the lead after the break then conceded a penalty late on which was converted before an 89th-minute winner.
The Reds played well for the majority of the game but were stung late on by Pompey who secured the league title.
Barnsley remain fifth, four points clear in the top six. They visit eighth-placed Blackpool on Saturday, who can still catch them, then host mid-table Northampton on the final day.
Neill Collins made two changes from Saturday’s home draw with Reading. Barry Cotter and full debutant Conor Grant came in for Corey O’Keeffe and the injured Jon Russell. Youngster Theo Chapman was on the bench with Luca Connell still out.
Barnsley started well and, after Adam Phillips volleyed a tough chance wide, Cole ended a 14-game goalless run with his 18th of the season. Grant found Cotter on the right of the box and his low scuffed cross was turned in from close range to totally silence the home crowd and delight the roughly 300 in the away end.
The equaliser came two minutes later when Tino Anjorin’s pass after a good move deflected to Kusini Yengi who smashed a powerful 15-yard finish past Liam Roberts.
The home crowd burst into life again then Anjorin then shot just wide from the left of the box. But Barnsley played well for the remainder of the half.
John McAtee’s weak 20-yard shot was easily saved after a good Jordan Williams pass. McAtee then had a much better chance as Herbie Kane’s superb pass put him clean through on goal but he was denied by goalkeeper Will Norris.
In the second half, Sean Raggett headed over from a corner then Yengi dragged wide from the edge of the box in space after Callum Lang flicked on a long pass.
Josh Earl’s long-range shot was easily saved but the Reds retook the lead just before the hour-mark when Nicky Cadden brilliantly sprinted deep into the home half and played through McAtee to supply a cool finish.
Portsmouth’s Lee Evans shot just over from 25 yards then Marlon Pack blazed over a free-kick from a similar distance.
The visitors were awarded a penalty when McAtee brought down substitute Christian Saydee on the right of the box and Colby Bishop netted from the spot.
Four minutes later Conor Shaughnessy headed in a right-wing corner from close range.
Substitute Fabio Jalo could not connect with a Cadden cross in injury-time.
Barnsley (3-5-2): Roberts; Williams, de Gevigney, Earl; Cotter (O’Keeffe 73mins), Phillips, Grant (McCart 88, Kane, Cadden; Cole, McAtee (Jalo 88)
Unused: Killip, Cosgrove, Marsh, Chapman.
Yellow cards: De Gevigney.
Goals: Cole 5, McAtee 59.
Portsmouth (4-2-3-1): Norris; Swanson (Rafferty 31mins), Shaughnessy, Raggett, Ogilvie; Evans (Saydee 80mins), Pack; Kamara, Anjorin (Lang HT), Whyte (Lane 61), Yengi (Bishop 61).
Unused: Macey, Moxon.
Yellow card: Lane.
Goals: Yengi 7, Bishop (pen) 85, Shaughnessy 89.
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