Having won 1-0 at Tranmere the previous week, Barnsley had retained the same back four and again they performed well to keep a clean sheet.
But the real Barnsley hero proved to be goalkeeper Nick Colgan, who made a string of superb saves, the highlight being a magnificent stoppage time save that denied Colchester substitute Gareth Williams.
Colchester, who had gone down bravely to Premiership champions Chelsea in the FA cup just seven days earlier started brightly at Oakwell and were the better side for the opening 20 minutes.
The long throws of Greg Halford were a real problem for the Barnsley defence and Richard Garcia should have taken advantage of one such delivery into the six yard box, but the Colchester striker was thwarted by Colgan's brave dive.
Barnsley also had their chances and Stephen McPhail was unlucky with a left foot shot that flashed just wide.
Colgan again caught the eye in the Barnsley goal with a fine fingertip save to keep out an edge of the box drive from Mark Yeates.
Barnsley finished the half strongly and Brian Howard should have scored when he set up by Paul Hayes but Colchester keeper Aidan Davison dived to his left and made a splendid stop.
Barnsley continued to press after the break and after winger Martin Devaney squandered a free header from 12 yards, the breakthrough finally arrived in the 73rd minute.
Devaney delivered the final left wing cross and although Howard's header was parried by Davidson, Howard made no mistake with the rebound.
Colchester' laid siege to the Barnsley goal in the dieing minuets but Colgan single handedly kept them at bay.