It means just a point from their final day clash with Tranmere would be enough to secure a play-off place.
They took control with two goals in four minutes just before half time as first Paul Hayes made it 20 for the season with a close-range finish after 36 minutes, and then skipper Cliff Byrne doubled the lead by drilling home an angled shot for just his third goal for the club.
As Cheltenham faded after a bright start it was Scunthorpe who dominated the second half, and Arsenal loanee Henri Lansbury made it 3-0 with some good footwork in the 67th minute.
But in stoppage time he was stretchered off with a suspected broken leg after a challenge from Yuri Berchiche.
The Cheltenham man received only a yellow card from referee Andy Hall but was dragged off the field by manager Martin Allen, leaving his side to finish with just 10 men.