Toulouse failed to move level on points with Ligue 1 leaders Paris St Germain and Marseille for the second successive week after a Chahir Belghazouani double inspired Ajaccio to a surprise victory.
Having slipped up at Bordeaux last weekend, coach Alain Casanova this week demanded a response from his side but it failed to materialise as they slumped to their third defeat of the season.
Despite being boosted by a fourth-minute own goal from Anthony Lippini, Toulouse trailed at half-time after goals from Frederic Sammaritano and Belghazouani.
Sigamary Diarra made it 3-1 before Aymen Abdennour gave Toulouse brief hope which was extinguished by Belghazouani's second of the night.
Toulouse could not have asked for a better start when Lippini diverted Etienne Didot's cross into his own net.
But Ajaccio levelled 11 minutes later as home keeper Ali Ahamada could only push Eduardo's shot into the path of Sammaritano, who made no mistake.
Ahamada saved well to keep out a Belghazouani effort on the half-hour mark but he was arguably at fault when the former France Under-21 international struck again four minutes later.
Ahamada's woeful clearance was seized on by Belghazouani and he prodded in at the second attempt.
Toulouse came close to an equaliser before the break with Yoann Poulard blocking Wissam Ben Yedder's powerful volley on the line while, after 51 minutes, Ajaccio goalkeeper Francisco Ochoa did well to deny Didot and shortly after Franck Tabanou.
But it was Ajaccio who scored next as Belghazouani turned provider to play in Diarra for a neat finish on the turn.
Abdennour scored following a goalmouth scramble to reduce the deficit after 72 minutes but Ajaccio had the final say as Ricardo Faty played in Belghazouani and he finished coolly.