The Iron's hopes of Championship survival were dealt a further blow at Wolverhampton Wanderers on Tuesday evening.
United went down to a 2-1 defeat in a story familiar to that acted out at Queens Park Rangers three days earlier.
Scunthorpe had a dream start when Andy Butler headed home from a Grant McCann corner, and looked fairly comfortable heading into the half-time break.
Just before the interval, McCann controversially had a goal disallowed for offside.
The play-off chasing hosts hit back after the break though, with Michael Gray firing home in spectacular fashion, and then a late headed effort from Neil Collins after a Matthew Jarvis cross sealed all three points for Mick McCarthy's men.
This was the Iron's game in hand on their relegation rivals and, as a result of the loss, they still sit six points adrift of safety - leaving it all to play for with seven matches remaining.
United made two changes from the side which lost at QPR over the weekend, as Paul Hayes and Izzy Iriekpen sat out at the expense of Ian Morris and Martin Paterson, who had returned from illness and injury respectively.
Scunthorpe also switched formation from 4-5-1 to 4-4-2, with Jack Cork deputising at right-back.
The match started brilliantly for Nigel Adkins' side, taking an early lead as Butler rose highest to bullet a header into the left corner after McCann's pinpoint corner. It was his second goal of the season, and the sort of strike he was netting regularly in League Two three years ago.
And they could have made it two just four minutes later when George Elokobi cleared off the goal-line after Paterson and Horsfield tried to convert a low Morris pass.
Wolves centre-back Darren Ward headed over the crossbar after a Darren Potter free-kick reached him, and Joe Murphy then gathered easily from a Gray cross.
Paterson came close just after with an effort from range which skimmed the top of Wayne Hennessey's net.
The hosts then had another chance when Sylvain Ebanks-Blake almost got on the end of Elokobi's cross, after a surging run forward from the defender.
Murphy was forced into action again with four minutes of the half remaining as Ebanks-Blake drew a save with his legs after a Matt Jarvis throughball.
And, in first-half stoppage time, Scunthorpe had the ball in the net once more - but McCann's close range effort was controversially flagged for offside.
The home side started the second period strongly, and levelled the scores on 57 minutes through ex-Sunderland man Gray.
Jarvis' free-kick could only be cleared to the edge of the area and there waiting was the left-sided player, whose fiercely struck half-volley flew past Murphy into the roof of the net.
Molineux was jumping, and Wolves continued to press for the win.
Butler forced a save from Hennessey after McCann's free-kick, while Ben May replaced Paterson as the visitors went with two target men up front, and Morris was replaced by Curtis Weston shortly after.
Ebanks-Blake hammered the crossbar on 80 minutes as he pounced on defensive indecision, and midfield man Seyi Olofinjana had a header cleared off the line.
Then came the suckerpunch three minutes later as Collins headed home a cross from Jarvis.
United tried to respond but lacked punch going forward. McCann drilled an effort wide from 20 yards, as the visitors attempted to force home an equaliser.
They had a great chance to double their advantage late on, when Andy Keogh's effort from close range was cleared off the line by Butler.
Collins' goal proved the match-winner for Wolves, who have now beaten Scunthorpe twice this season.
The Iron will be hoping their luck can change when Ipswich Town head to Glanford Park on Saturday.
