Paul Cook's men fell to an embarrassing 3-1 loss at relegation-threatened Accrington Stanley, who spent almost an hour down to 10 men, on Saturday.
That defeat, which came after a goalless home draw against struggling Portsmouth, means Chesterfield are just a point ahead of Scunthorpe United and Rochdale - who sit second and third respectively.
And with the latter holding a game in hand, Chesterfield's trip to AFC Wimbledon on Tuesday takes on added importance.
Scunthorpe meet Southend United and Rochdale travel to bottom-of-the-table Torquay United.
Southend, currently occupying the final play-off place, have plenty to play for at Glanford Park given they are a mere two points ahead of eighth-placed Plymouth Argyle, who have played a game less.
Argyle travel to face a Wycombe Wanderers side that beat fellow play-off-chasers Hartlepool United 2-1 last time out to extend their unbeaten run to six games.
Wycombe will hope Hartlepool can do them a favour on Tuesday, though, with Colin Cooper's men heading to Accrington.
Stanley, now six points clear of the drop zone in 22nd, have a player brimming with confidence in Lee Molyneux after his hat-trick earned that aforementioned triumph over Chesterfield.
The other three sides in the top seven, Burton Albion, Fleetwood Town and Oxford United, are aiming to solidify their positions.
Burton and Fleetwood are on the road, facing Portsmouth and Bury respectively, while Oxford host Cheltenham Town.
Should they slip up, York City and Newport County, who like Plymouth and Hartlepool are just outside the play-off spots, will aim to take full advantage.
York, unbeaten in six league outings, face a trip to Mansfield Town, while Newport head to Morecambe on the back of a five-game winless run.
Tuesday's action is rounded off by Bristol Rovers facing Dagenham & Redbridge and Exeter City tackling Northampton Town.
