Trinity were thumped 5-0 at home by Farsley Celtic on Tuesday night, just 10 days after going down 4-1 to Warrington Town in their previous home game.
The Holy Blues have won just two of their 11 home games, resulting in a drop off in attendances.
Club captain Liam King, who only this week opted to cancel his playing contract due to injury problems, will take temporary charge.
Gainsborough currently sit in sixth place in the EVO-STIK League Premier Division, four points off the play-off places but having played a game more.
Keep up-to-date with our exclusive email newsletters.
Subscribe