Craig Mahon returned from injury in style - hitting home a dramatic winner to complete a superb turnaround at Haig Avenue.
Louis Almond gave the hosts a lead they held until the 79th minute - before Michael Kay and Mahon strikes bagged the three points for Steve Burr's men.
Holmes was the first Sandgrounder to test Jon Worsnop - with a 20 yard free kick - before John Rooney volleyed over from 25 yards.
Chester took control in the opening quarter of an hour though; with Jordan Chapell firing over - before Johnny Hunt sent a volley wide of the right hand post.
Ross Hannah then headed straight at Andy Coughlin - following a dangerous cross from Ben Heneghan.
Louis Almond sent out a warning sign - almost latching onto a pass - before giving the hosts the lead on 36 minutes.
The forward was allowed far too much space on the edge of the box - enabling him to check onto his right foot before firing home.
The Blues did press forward in search of an equaliser in the second half - but struggled to break down a stubborn Southport defence.
That was until eleven minutes from time.
The home defence failed to clear a Rooney corner - allowing defender Michael Kay to smash the ball into the back of the net.
Chester had the momentum behind them - and got their noses in front just five minutes after the leveller.
Two subs combined - Craig Hobson heading a loose ball across the box; allowing Mahon to slot home from close range - securing a dramatic away win.
Southport: Coughlin, Nolan, Jones (Thompson 66), Foster, Davies, Nolan, Wright, Joyce, Almond, Phenix, Stockton, Bishop (Rutherford 77).
Subs Not Used: Viscosi, Allen, Blakeman.
Chester: Worsnop, Higgins (Richards 61), Sharps, Kay, Hunt, Heneghan, Shaw, Rooney, Chapell (Hobson 78), Thomson (Mahon 61), Hannah.
Subs Not Used: Forth, Hughes.
Scorers: Almond 36; Kay 79, Mahon 84.
Attendance: 1,326 (407 away).
Referee: Adrian Holmes.