Gosport slipped out of the play-off places after suffering their third loss on the trot in a 1-0 defeat at Hitchin Town.
Having gone 23 league matches unbeaten, Borough were out-of-sorts in Hertfordshire and went down to a John Frendo winner.
Barwell’s win at Bideford sees Alex Pike’s men drop to sixth, although Bedford Town did Gosport a favour by beating fellow hopefuls Weymouth 4-0.
Gosport, already missing the injured Justin Bennett, added Brett Poate to their list after the former Hawk failed a late fitness test, with Ellis Martin taking his place.
The hosts pushed from the start and keeper Nathan Ashmore had to be at his best to punch clear, with striker Lewis Toomey finding himself clear and ready to line up a finish.
Martin showed his worth with a fine clearance, as Borough’s defence came under pressure.
Dan Woodward then went clear to set up Dan Wooden whose volley flew just wide.
At the other end, Ryan Woodford came to Borough’s rescue, getting his head to a vicious, curling shot from Frendo that was destined for the roof of the net.
Slowly but surely Gosport began to sparkle as Sammy Igoe took a Rory Williams pass in his stride to bring a flying save from Martin Bennett at his near post.
Borough fans began to celebrate what they thought was the opener when Williams powered in a shot, only to find the ball had hit the side-netting.
Just before the interval, it took a high challenge - and subsequent booking - for Dan Smith to deny Callum Donnelly a clear run at goal as Borough’s defence, for once, failed to deal with a long, route-one clearance.
More Hitchin pressure had Borough struggling to deal with the pace of Mike Noone, with Luke King getting a yellow card for bringing his run along the line to an end.
Toomey went close for the hosts after Ashmore failed to take a harmless cross but King was there to clear the danger.
A series of corners followed for the hosts and one was finished clinically by Frendo, who met Noone’s cross at the near post to volley past Ashmore.
And before Borough regained their composure, Frendo should have doubled the advantage after being sent clear but his half-hit shot was scooped up by Ashmore.
Gosport’s fate was sealed as Hitchin pulled everyone back into defence in the latter stages to safeguard their lead.
Gosport chairman Mark Hook said: ‘We never got out of the starting blocks. The lads were lethargic and never looked like recovering once they fell behind.
‘But we live in hope. The season has some way to go so let’s hope fortune shines on us and we can still make the play-offs.’
Southern League premier division
HITCHIN TOWN 1 (Frendo)
GOSPORT BOROUGH 0
Hitchin Town: Bennett, Legg, Bickerstaff, Stewart, Gilbert, Lee, Donnelly, Gregson, Toomey, Frendo, Noone. Subs: Webb, Remy, McGeorge, King, Burke.
Gosport Borough: Ashmore, King, Martin, Cook, Woodford, Dunford, Igoe, D Smith, Wooden, Williams, Woodward. Subs: Brown, Ender, S Smith, Molyneaux, Wilde.