Stranraer forward Darryl Duffy piled the pressure on Hamilton head coach Brian Rice by consigning Accies to an early Betfred Cup exit.
Duffy's 81st-minute header secured a 2-1 victory for the hosts at Stair Park and added to Rice's misery following his side's 8-0 defeat by Rangers on Sunday.
Rice had been confronted by a number of angry fans when he and his squad returned from Ibrox and the supporters' ire will have been exacerbated by a defeat against a team sitting one point off the bottom of League Two.
Rice made six changes and handed debuts to former Motherwell and Hartlepool winger Nathan Thomas and ex-Coventry and Exeter defender Aaron Martin.
But Thomas Orr had threatened on several occasions for Stranraer before firing home from inside the box in the 34th minute.
Accies equalised in the 70th minute when Hakeem Odoffin headed home from close range following Kyle Munro's corner.
But the hosts regained the lead when Duffy headed home Hilton's left-wing cross and held on to move a point behind Group F leaders Annan, who beat Hamilton last month.
Accies sit on three points ahead of their final game at home to Albion Rovers on Saturday and cannot amass enough points to reach the second round.