The Tykes bounced back, just like they had done at Hull on Tuesday night, and this time it was Colchester who fell victim to a strong second-half performance.
The U's took the lead on 42 minutes when Greg Halford steamed in to head home a cross from Jamie Cureton at point-blank range.
But Barnsley came storming back in the second period and goals from Marc Richards and Brian Howard ensured a second successive away win for Andy Ritchie's men.
It was also a special occasion for Mr Poll, who was officiating in his first competitive match since making a much publicised blunder in the World Cup.
Mr Poll made a name for himself for the wrong reason by showing a Croatian player three yellow cards before sending him off in the 2-2 draw with Australia.
All eyes were on Mr Poll this afternoon, but he didn't put a foot wrong at Layer Road, one amusing moment came when he booked his first player Kemal Izzet in the 17th minute.
The Layer Road crowd responded with a chant of "You only need two more", implying that the Hertfordshire referee might even repeat his error from the summer.
Mr Poll took it in good humour, with a wry smile.
Both sides created chances galore before Halford nodded home his first of the season. It was only the club's second of the campaign.
But it didn't last for the Essex side. Strikers Richards, who missed several chances in the second half, headed home a Michael McIndoe cross in the 57th minute.
And a long-range bullet from Howard inflicted a third straight defeat on Colchester with 12 minutes remaining.
U's keeper Aidan Davison had no chance as Howard crashed home a shot from 25 yards out.
Barnsley had bounced back from two down to win 3-2 at Hull in midweek, but manager Ritchie will be wanting his side to take the initiative rather than keep coming from behind too often.
As for the U's, they are without a point in three games and they face a daunting trip to West Brom in their next match.