First-half goals from Alex Mowatt and Cameron McGeehan secured victory for the Tykes, who took the lead in dramatic fashion in the 16th minute.
Former Reds winger Adam Hammill was taken down going for a loose ball inside the box by Mamadou Thiam, Barnsley countered and Mowatt took aim from 40 yards and beat goalkeeper Jak Alnwick, who was short-sighted by the low sun.
Barnsley doubled their advantage four minutes before half-time as Thiam tried his luck from distance, the ball took a deflection and looped onto the head of McGeehan, who guided the ball past Alnwick.
Scunthorpe created their best chance in first-half stoppage time as Lee Novak met a low cross and forced keeper Adam Davies into a smart save at point-blank range, tipping the ball onto the roof of his net.
Barnsley dominated the second half but moved up to second in the table despite being unable to force a third goal.