A Ryan Loft goal at the end of first half stoppage time proved too much for Town to come back from as Simon Weaver's side fell to defeat at Glanford Park.
Loft had created the opener for Devarn Green after just four minutes, but Josh March levelled from the penalty spot mid way through the half.
The decisive minute arrived in the third minute of first half stoppage time as Loft latched onto a clearance and beat James Belshaw, though Lewis Spence did add to the hosts' tally with a late long range effort.
Weaver named the same squad that picked up three points against Carlisle on Tuesday night, with Captain Josh Falkingham partnering George Thomson in the heart of midfield.
Despite having not played since January, the hosts made the better start with Green finishing Loft's low cross at the far post to put his side ahead after just four minutes.
Town started to apply pressure after the early setback, with multiple set pieces delivered into the Scunthorpe box and Aaron Martin heading off target after 18 minutes.
It was almost two for the hosts after Loft had put the ball in the back of the net on 25 minutes, only to be flagged offside by the linesman.
Within seconds of the disallowed goal Town were level as March continued where he left off against Carlisle, winning and converting a penalty kick to claim his fifth goal for the club.
Town looked to be finishing the half stronger with Josh McPake almost finding the top corner with a deflected effort, but his side would go in behind at the break.
In the third minute of stoppage time, Loft latched onto a clearance to race through on goal from where he was able to beat Belshaw and put his side back in the lead.
Town struggled to recapture the pressure applied to their opponents in the first half, with no real sights of goal, prompting Weaver to throw Mark Beck into the action with 20 to play.
McPake continued to look lively and pose a threat to the Iron goal, but chances weren't falling Town's way.
With 90 minutes on the clock, substitute Spence sealed the Scunthorpe victory, rifling home from long range and leaving Belshaw no chance.
Town: Belshaw, Falkingam, Smith, Burrell, Thomson, Martin (Beck 70), Hall, Francis, March, McPake, Power (Muldoon 88)
Subs not used: Lawlor, Kiernan, Kerry, Minter
Goals: March 27 (p)
Scunthorpe: Howard, Clarke, Taft, Onariase (Bedeau 77), Gilliead, Loft (Hallam 84), Eisa, Brown, Green (Van Veen 59), Beestin, Karacan (Spence 77)
Subs not used: Watson, Rowe, O'Malley
Goals: Green 4, Loft 45+3, Spence 90
Referee: John Busby