Manager Alex Dyer criticised a "shocking decision" from the referee as Kilmarnock were downed by two goals in the final two minutes from Livingston.
Jaze Kabia and Scott Pittman scored at the death as late pressure paid off to move Livi to win five points of fourth with a game in hand.
But Dyer was unhappy after captain Stuart Findlay was forced off injured during the defeat.
"He didn't get booked or anything, it was a foul," Dyer told BBC Scotland.
"A shocking decision from the ref. He [Findlay] came in at half-time and realised he couldn't come out second half, his ankle was sore and he's twisted his hamstring as well, so that's a double blow for us."
Three days after reaching the League Cup final, David Martindale's Livingston made it 12 games unbeaten in all competitions.
Kilmarnock drop to eight in the table after back-to-back defeats.
Dyer had been "proud" and "overwhelmed" when Kilmarnock moved up to fifth in the table with a 3-1 win on their last visit to West Lothian in October. However, by the time Livingston exacted revenge at Rugby Park in December, Dyer was fearing for his job as the two sides' fortunes switched.
This time, Kilmarnock arrived missing key players and very much the underdogs and their hosts, buoyed by defeating St Mirren in the League Cup semi-final three days earlier, were out for blood from the start.
Alan Forrest forced goalkeeper Danny Rogers into a save after Aaron McGowan had failed to deal with a through ball, shortly before Jon Guthrie headed a Josh Mullin corner off the bar.
They went close again when Gavin Reilly pounced on a Zeno Ibsen Rossi error, cut-back to Pittman, whose shot was charged down.
In the aftermath, Kilmarnock defender Stuart Findlay was left limping and, although he battled on until half-time, he eventually had to make way for Ross Millen.
Debutant Kilmarnock striker George Oakley ballooned a shot over the bar from close range in a rare venture into the opposition box from the visitors.
Guthrie headed over again for Livingston and Forrest couldn't keep his effort down either as Livingston grew as a force the longer the second half went on.
By the final 10 minutes, it was all the hosts, but Kilmarnock were defending in numbers and looked set to secure a hard-earned point. However, with full-time in touching distance, Livingston struck two late blows.
Neat play from Craig Sibbald on the right opened up Kilmarnock, the ball broke to Kabia at the back post and the forward fired low into the corner, with the aid of a deflection off Millen, beyond Rogers.
Moments later and a long ball caused angst in the Kilmarnock backline. As they failed to clear, Pittman stole in behind the defence and poked the ball under Rogers.
Man of the match - Alan Forrest
What did we learn?
With Aberdeen and Hibernian dropping points, Livingston had an opportunity to put pressure on the sides above them with the Europa League spots now well within their sights.
They made four changes from the Hampden semi-final and didn't play anywhere near their best football, but through shear perseverance they got the job done.
One loss will be Ciaron Brown, who played the 90 minutes for Livingston but is now set to return to his parent club, Cardiff City.
Kilmarnock arrived with key players missing and the loss of Findlay added to their woes. They offered little threat, while defensively often it was unforced errors that gifted Livingston their chances.
What did they say?
Livingston manager David Martindale: "I think Kilmarnock will be getting sick of us and late goals. But I think we more than deserved the three points.
"I passed the fit and proper test this week so that was part of my pre-match speech: 'Don't let me down, don't let me down'."
Kilmarnock manager Alex Dyer: "We should have see the game out, definitely. We can't keep saying it, all our games are close - one goal or two goals max. Sometimes you have to look at it and see if it's bad luck or just bad play sometimes.
"During the season people have been saying how bad our season had been, but we've always been ahead of certain teams and people are saying we haven't been having a good season."
Livingston are at home to Aberdeen on Saturday (15:00 GMT), while Kilmarnock host St Johnstone (15:00).