It looked as though Juve were going to preserve their four-point advantage at the summit with four games to play and edge closer to a seventh successive Scudetto only for Koulibaly to throw the race wide open.
Compounding Juve's last-gasp defeat is the fact Massimiliano Allegri's men arguably have the tougher run-in of the two sides, with the Derby d'Italia away to Inter coming up next Saturday, and a clash with Champions League semi-finalists Roma awaiting in the penultimate round of fixtures.
Napoli, meanwhile, seeking to win a first Scudetto since 1989-90, when they were inspired to a second title in four seasons by the great Diego Maradona, visit Fiorentina next weekend hopeful of seizing on any further slip-ups from the leaders.
Napoli enjoyed an early sight of goal when Lorenzo Insigne fired wide in the first minute, and the passage of play proved costly for Juventus, as Giorgio Chiellini sustained an injury that ultimately forced him off 10 minutes later, having failed to convince in his attempts to shake off what appeared to be a muscle problem.
With full-back Stephan Lichtsteiner introduced and Benedikt Howedes shifting to centre-back, Juve went close for the first time in the 17th minute, Miralem Pjanic's deflected free-kick wrongfooting Pepe Reina and striking the far post, leaving the Napoli goalkeeper stranded.
His counterpart Gianluigi Buffon had to be alert to tip over a dipping cross-cum-shot from Mario Rui, who then slid in Marek Hamsik for the best chance of the first half in the 24th minute, only for the Napoli captain to drag his attempt across the face of goal and wide.
Insigne had the ball in the back of the net seven minutes before half-time, but Lichtsteiner smartly stepped up to catch his opponent offside.
Allegri responded to his team's underwhelming first-half showing by withdrawing Paulo Dybala in favour of Juan Cuadrado, while Maurizio Sarri introduced the fit-again and in-form Arkadiusz Milik a minute past the hour mark, taking off the relatively ineffectual Dries Mertens.
Substitute Piotr Zielinski tested Buffon from long range with seven minutes to go in what had been a tense second half, and the goalkeeper also had to claw away an awkward Jose Callejon delivery.
From the Spaniard's subsequent corner, Napoli grabbed the all-important goal, Koulibaly rising above the Juve backline to head home a powerful winner, the final whistle sparking wild celebrations among the visitors and ensuring a thrilling conclusion to the title race.
