Adam Phillips' strike in the first half proved the difference, and despite an onslaught from Pompey in the second half they couldn’t get the goals they needed to turn the game around.
Cowley made a surprise change in the Portsmouth line up, with Sean Raggett on the bench having started 43 games this campaign – Jack Whatmough coming in after serving a suspension.
The two teams played out a 3-3 thriller just ten days earlier, and Pompey had a good chance of going ahead within five minutes when John Marquis was played through down the left by Ronan Curtis, but he couldn’t lift the ball over the advancing Toby Savin.
Stanley strike first
But it was Accrington who broke the deadlock 18 minutes in. Harvey Rodgers found Phillips in space inside the area, who was afforded plenty of time before he sent a crashing effort past Craig MacGillivray from 12 yards which went in off the inside of the post.
Phillips then almost doubled his and Stanley’s tally with a bullet free-kick from over 30 yards out, and MacGillivray did well to get across to save low to his right.
Portsmouth were fortunate not to fall further behind ten minutes before the break when Rodgers lost his marker and had a free header from Sean McConville’s corner, but his header was blocked by Marquis.
Accrington were well worth their lead, but Pompey almost drew level in first half added time when Curtis flashed a ball across the face of goal, but it went wide of the far post.
And the hosts had a huge sigh of relief before the break when Rodgers met Phillips’ free-kick and powered a header into the top corner, but the flag went up for offside against the Accrington midfielder.
Pompey turn the screw
Portsmouth showed more urgency in the early stages of the second half, and almost drew level through a superb solo effort from Curtis. Picking the ball up on halfway, he drove into the space that opened up for him but his low drive from 25 yards was tipped around the post by Savin.
Harness then broke into the box down the right before shifting the ball across to Curtis, but this time he leaned back too much and his first time effort went over the top.
The Portsmouth side who came out for the second half looked so much more determined, with Curtis again at the heart of the action as the hosts went close again. Pompey’s #11 cut in from the left and this time played through Marquis, but Savin was out well to save.
Cowley made a treble change with just under an hour gone – knowing his team needed to come from behind to take all three points if they were to make the play-offs. On came George Byers, Harvey White and Jordy Hiwula in place of Ben Close, Callum Johnson and Paul Downing, with Pompey changing their shape to 3-4-3 in the search of two goals.
And Byers almost made the desired impact. Found by White, he skipped past two challenges but his low drive from 18 yards was well held by the diving Savin.
Time running out
With fifteen minutes to go Harness whipped in a corner from the right, but Tom Naylor couldn’t direct his header on target as Pompey were running out of time.
Harness then did well to find sub Charlie Daniels, who broke through on goal but Savin was equal to his effort with Pompey throwing everything forwards.
But the hosts couldn’t break down Accrington, meaning they miss out on the play-offs after Oxford United’s victory over Burton Albion.