Danny Wright’s fourth goal for Harriers was not enough as Gary Whild's men left the Silverlake Stadium empty-handed.
Ben Strevens and James Constable hit back for Eastleigh as they confirmed their place in the Vanarama Conference play-offs.
Harriers, meanwhile, finish the 2014/15 season in 16th.
Whild made two changes to the side which drew 1-1 against Barnet last Saturday, and seven to the XI which started the Worcestershire Senior Cup victory midweek.
returned after recovering from a groin problem, and Luke Maxwell kept his place after scoring his first senior goal against Worcester City on Tuesday.
They came in for Jake Green and Nat Kelly, both of whom started in midfield last week but dropped to the bench today.
Eastleigh, on the other hand, made one alteration to the team which claimed victory at Lincoln City, as Craig Stanley replaced Harry Pell.
Harriers required just 50 seconds to strike through Wright, who took advantage of an error of judgement by Ross Flitney.
The Spitfires ‘keeper had come charging out of the box to try and claim the ball but missed it, allowing Wright to claim possession.
A swift advance towards goal was rounded off with a delightful finish by Wright, a low shot from an incredibly tight angle which sneaked around the near post, despite the ultimately futile efforts of Joe Partington on the line.
However, the hosts stumped up the pressure gradually after conceding, and struck in the second half through Strevens and Constable.
Seven minutes after the interval, Strevens equalised for Eastleigh, poking the ball home after a Jai Reason corner ended in an almighty goalmouth scramble.
And just over 20 minutes later, the Spitfires had turned the game in their favour, although it came in controversial circumstances after they were awarded a dubious free-kick by referee Lee Swabey.
From the set-piece, a venomous strike from outside the area by Reason, a common theme throughout the match, crashed off the right-hand post, and the lurking Constable was well-placed to get to the rebound before Danny Lewis
and slot home.
Harriers’ early goal was the stand-out moment in a first half well-contested between the two sides but lacking in clear-cut opportunities in front of goal.
Kevin Nicholson almost doubled Harriers’ lead minutes after Wright’s goal, the left-back’s long-distance free-kick on the left almost sneaking underneath the crossbar, forcing Flitney to tip it over.
Eastleigh took a while to get into their stride, as Craig McAllister and Jack Midson nodded wide inside the first half hour.
The hosts initially threatened mainly from long-distance efforts, as Strevens and Reason stung Lewis’ palms with fierce shots from outside the area as the interval approached.
Reason’s effort six minutes before half time could only be parried as far as the lurking Midson, but his follow-up header was flagged offside, and tipped clear by Lewis regardless.
Maxwell and Midson had shots on target as the first half fizzled out, the latter’s a little more firm than the Harriers Academy graduate’s cross-turned-shot, but despite the simmerings of an Eastleigh resurgence, Harriers took their one-goal lead into half-time.
After emerging from the interval, the Spitfires began as they meant to go on and started to increase the pressure.
Lewis was forced into a magnificent point-blank save three minutes into the second half, as former Harriers frontman Constable powered a header towards goal from Midson’s left-sided cross.
Wright almost increased Harriers’ advantage when, fed by Lee Hughes, he hooked an effort over Flitney but not past Dan Spence, who cleared the danger as the ball headed towards goal.
Eastleigh then put pay to Harriers’ hopes of taking something from their trip to the south with two goals in 20 minutes, with Whild introducing Ahmed Obeng and Kelly before the hosts scored the winner.
Constable continued to pester as the game drew to a close, while substitute Deon Burton also had a couple of chances of his own, but they had already done enough to earn the win.
After the game, Harriers' players and staff approached the away end to applaud the 148 fantastic travelling fans who were vocal throughout. Thanks for your support during 2014/15.