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The visitors, currently pushing for promotion from the Second Division, opened strongly with Tony Kay directing a third minute header wide.
Two minutes later Steve Hayward's free-kick caused no problems for the Iron's rearguard.
United shook the Tykes when they took a 14th minute lead as Steve Torpey rose high above the visitors defence to flick home a header from a free-kick which had been hammered in by Lee Ridley.
Torpey proved to be a pivotal figure as the Second Division side began to force the pace in an attempt to level the match, managing to get in several telling headers to clear attacks.
A point-blank shot from Jacob Burns was well saved by Tommy Evans, continuing his tremendous FA Cup form this season, and he was on hand again moments alter to block an effort from Isaiah Rankin.
Scunthorpe almost grabbed a second as the second half kicked off, but a superb tackle from Craig Ireland halted Steven MacLean in full flight.
Ireland then found himself at the other end and he turned Hayward's cross onto the woodwork.
That was almost the end for the Tykes as Scunthorpe hit them on the counter, makeshift striker Jamie McCombe curled home a superb right-foot shot from deep on the right in the 74th minute to take the wind out of Barnsley's sails.