Last season's League One playoff winners scored a goal in either half to pick up the victory, following on from their triumph by the same scoreline over Stalybridge Celtic on Saturday.
Adam Hammill had the only shot on target of the opening period, but it was a tame effort and hosts Guiseley were the first to seriously threaten when Michael Rankine fired over from outside the area after 16 minutes.
The Tykes were in front just six minutes later, however, when Hammill's cross was met by James Carvell, who poked it home to break the deadlock.
Carvell threatened again with 10 minutes remaining of the half, but this time he curled his effort just wide after collecting the ball and driving at the defence.
There were no further goals in the first half, but it took Barnsley just seven minutes after the restart to double their advantage when Shaun Tuton and Kayden Jackson combined to pick out Stefan Payne, who made no mistake with his finish.
The second half proved to be an uneventful affair with chances at a premium, and in the end Barnsley saw out the match comfortably to ease to the victory, making it six wins in a row including the tail end of last season.