Napoli's Coppa Italia defence ended when they gave up the lead to lose 2-1 to Bologna on Wednesday, while Lazio advanced on penalties.
Napoli defeated Serie A champions Juventus 2-0 to win Italy's national cup competition in May, courtesy of goals from Edinson Cavani and Marek Hamsik.
It was key man Cavani who scored to give Napoli the lead 12 minutes in at the Stadio San Paolo, only to see Cristian Pasquato level with seven minutes remaining in the half.
With neither team able to force a breakthrough in the second period, the tie looked set for extra-time, until Panagiotis Kone netted in the 91st minute to snatch an upset win for Bologna.
Elsewhere, Lazio needed a 95th-minute strike from Michael Ciani to force their match against Siena into extra-time.
The visitors had led following Lorik Cana's own-goal on 56 minutes at the Stadio Olimpico, but it was Lazio who came out on top, 4-1 on penalties following a goal-less additional 30 minutes.
Finally, Borja Valero's lone strike was enough for Fiorentina to beat Udinese 1-0 away from home.