The managerless Honest Men defeated the Lowland League side 3-0 at Dumbarton, watched by Steven Gerrard, Ross Wilson and Gary McAllister.
However Ayr have now been summoned to an SPFL disciplinary hearing after being accused of breaching competition rules.
After 81 minutes of Tuesday night’s game, caretaker manager Jim Duffy sent two substitutes – Kinlay Bilham and Alex Jeanes – into the action. Both are currently on temporary transfers to Irvine Meadow XI and Girvan in the West of Scotland league which would render them ineligible.
If found guilty of the breach, the Honest Men’s punishments could include reversing the result and ejection for the Championship side.
Earlier this season in the Premier Sports Cup, the SPFL’s other knock-out competition, Kilmarnock fielded a suspended player – Daniel Armstrong – against East Kilbride and were hit with a 3-0 defeat as punishment.