Town's ten-match unbeaten run was brought to an end at Silverlake Stadium as Eastleigh came back from behind to send Simon Weaver's side home empty handed.
Despite Brendan Kiernan's sixth goal of the season giving Town the lead, Scott Rendell's double sent the hosts into half time ahead.
Tom Bearwish added a third in the second half, with Jack Muldoon firing back for Town, only for Rendell to find himself one on one seconds later from where he was able to complete his hat-trick and inflict a first defeat since September.
Weaver named the same starting XI that overcame Barnet at The CNG Stadium on Tuesday night, with Sam Jones replacing the suspended Jon Stead on the bench.
The first chance of the game went the way of the hosts when, after George Smith had pulled back Sam Smart on the Eastleigh right, Rob Atkinson met Jack Payne's free kick, but headed wide of the target at the far post.
From the resulting goal kick, Muldoon was able to get hold of the ball and find the run from Kiernan inside from the right wing, creating the chance for his teammate to slide the ball past Max Stryjek for his sixth goal of the season.
Eastleigh responded well to falling behind, applying pressure to the Town goal, though Kiernan was well placed to hook away the effort of Rendell who had stabbed towards goal from a corner.
James Belshaw then had to be alert to keep out a bobbling Jack Payne free kick that had looked like nestling in the bottom corner.
With ten minutes until the interval, Eastleigh were awarded a penalty when Ryan Fallowfield brought down the lively Sam Smart, allowing Rendell to equalise from the spot.
Six minutes later and the combination of Smart and Rendell provided the goods again as a tantalising cross from the right was powerfully headed past Belshaw by Rendell to put his side ahead at the break.
Eastleigh doubled their lead 12 minutes after the restart, George Smith thought he had cleared the danger after a lapse of concentration almost let Eastleigh in, but Bearwish was first to the loose ball and squeezed his finish through the legs of Belshaw.
Town almost provided an instant reply, but Diamond's fierce low drive deflected over the bar.
The pressure on the Town goal was only momentarily eased though as Warren Burrell was required to produce an acrobatic overhead kick to keep the ball from crossing the line and Eastleigh finding their fourth.
Jack Muldoon thought he had given Town hope in the 69th minute as he raced through on goal and managed to beat Stryjek, despite seeing his original finish blocked.
The hosts restored their lead in seconds though, Rendell getting on the end of a short back pass that left him one-on-one with Belshaw and able to get the better of his opponent.
With nine minutes to play, Fallowfield bombed forward down the right and cut back to Muldoon, only for Stryjek to deny the Town striker a second goal of the afternoon.
Despite piling pressure on the Eastleigh goal, neither George Thomson, Jack Emmett or Burrell could find a way past an inspired Stryjek in the closing stages.
Harrogate Town: Belshaw, Fallowfield, G Smith (Taylor 75), Falkingham, Burrell, Beck, Kiernan (Thomson 66), Bradley (Emmett 59), Muldoon, W Smith, Diamond
Subs not used: Jones, Cracknell
Goals: Kiernan 9, Muldoon 69
Booked: Falkingham, Fallowfield
Eastleigh: Stryjek, Smart, Atkinson, Boyce, Seaman, Miley, Hollands, Payne, Bearwish, Barnett (Williamson 83), Rendell
Subs not used: Flitney, Scorey, Beale, Waterfield
Goals: Rendell 36 (p), 41, 70, Bearwish 57
Referee: A Quelch
Attendance: 1592 (21 away)