A debut goal from Stoke loanee James Alabi wasn't enough for Brian Laws' side after they had conceded two first half goals courtesy of James Poole and Jonathan Franks.
The Iron made two changes to the team which started against Yeovil last week. Eddie Nolan and David Mirfin came into the defence to replace Callum Kennedy and captain Paul Reid.
Kennedy picked up a hamstring injury in the previous fixture while Reid was suffering from illness, so Michael Collins deputised as skipper in his absence.
Sam Slocombe started in goal, with a backline in front of him consisting of Eddie Nolan, Niall Canavan, David Mirfin and Tom Newey.
The midfield four was made up of Mark Duffy, Josh Walker, Michael Collins and Jimmy Ryan while Akpo Sodje and Karl Hawley starter upfront.
Recent loan signing James Alabi was named amongst the substitutes along with Connor Jennings, who returned at the beginning of the week following his loan spell at Stockport.
The first real chance came in the 7th minute, when Jimmy Ryan picked up the ball after great work from Eddie Nolan. He hit a shot from distance which was parried out of danger by Flinders.
There was nothing of any real purpose until seven minutes later when a flashed ball across the Scunthorpe area avoided everyone until being headed clear by Nolan.
In the 18th minute, Hartlepool took the lead. A cross was swung in from wide and met by Humphreys on the edge of the area. He headed to Wyke and the ball was held up well in the 18-yard-box before being laid off to Poole who rifled home.
The Iron looked to get straight back into the game, but after good work between Hawley and Sodje, the Nigerian striker smashed his shot high and wide.
Despite the goal, Brian Laws' men were unable to create any clear cut chances as Hartlepool's defence remained strong.
With ten minutes of the half remaining, the away side doubled their lead. The ball was lost by the Iron in the middle of midfield, and when it eventually found Franks, he ghosted past a number of defenders and slotted the ball into the corner of Slocombe's net.
Just before the interval, the visitors almost extended their lead when a corner kick found Peter Hartley, but his header was deflected just over the bar.
Scunthorpe went in at half time two goals down after what had been a largely uneventful game so far.
Brian Laws made an attacking substitution during the break when he replaced Josh Walker with Andy Barcham.
50 minutes in, a backpass from Canavan was sliced by Slocombe to Poole. He drove into the area and crossed but Newey got back in time to intercept before it reached Wyke.
Then, the Iron were unable to clear their lines after a stint of head tennis with the opposition. All of a sudden, Wyke struck a superb long range volley which thundered against the crossbar.
Just after the 60 minute mark, a long kick downfield from Flinders found Poole, he turned and hit a dipping effort which Slocombe had to tip wide.
Six minutes later, the home fans were able to get their first glimpse of new loan signing James Alabi, as he replaced Akpo Sodje.
Scunthorpe certainly began to come into the game as the half went on and looked much more dangerous on the attack.
Meanwhile, John Hughes' side continued to look for the pace of Franks on the counter attack, whose pace was causing a lot of problems.
In the 76th minute, the Iron pulled a goal back through substitute James Alabi. Hawley's pass out wide found Duffy, and after some neat footwork, he floated a cross which was headed into the top corner by the 18-year-old.
The home side continued to pile on the pressure in order to find a crucial equaliser. With five minutes stoppage time at the end of the game, Niall Canavan was thrown upfront as a makeshift striker.
With the very last kick of the game, Jimmy Ryan's long range bullet of a strike flew just wide of the left post and the score finished 2-1.
Scunthorpe United: Slocombe, Nolan, Mirfin, Canavan, Newey; Duffy, Walker (Barcham 45), Collins (c), Walker, Ryan; Hawley, Sodje (Alabi 66).
Substitutes: Severn, Ribeiro, Grella, Jennings, Godden.
Hartlepool United: Flinders, Austin, Collins, Hartley (c), Horwood; Franks (James 80), Walton, Baldwin, Humphreys; Poole (Monkhouse 68), Wyke (Howard 60).
Substitutes: McHugh, Luscombe, Rutherford, Holden, James.
Attendance: 3,434 (359 away).
Iron Bar man of the match: James Alabi.