Early headers from Dan Ndoye and Stefan Posch were enough for Thiago Motta's Bologna to move third, above Juventus, and go seven points ahead of sixth-placed Roma, with a top-five finish enough for a spot in Europe's top club competition.
Roma are at fifth-placed Atalanta -- who have a game in hand -- on Sunday night with the pair level on 60 points, and anything but a win for the capital club will guarantee Bologna a place in the Champions League.
Bologna played one match in the old European Cup in 1964, the same year they won the last of their seven league titles, but have never featured in the modern Champions League.
"More than the Champions League I'm happy about the squad who once again put in a really good performance against a good team," Motta said to DAZN.
"I'm putting all my energy into enjoying the moment because the boys were great."
The only negative note on a great evening for Bologna was star forward Joshua Zirkzee limping off in tears with what looked like a thigh injury.
Dutchman Zirkzee, who is yet to claim a senior cap for his country with Euro 2024 around the corner, was consoled by team-mates in the Bologna dug-out.
Napoli were loudly booed off at half-time and at the final whistle by livid fans who have watched their team slump from Scudetto winners last season to eighth this term.
The hosts had a chance to get back into the game when Victor Osimhen won a soft penalty in the 20th minute, but Federico Ravaglia pushed away Matteo Politano's poor spot-kick.
There was more fan discontent in AC Milan's 5-1 thumping of Cagliari -- who stay three points above the relegation zone -- in their penultimate home game of the campaign.
Supporters at the San Siro continued the silent protest they started at last weekend's home draw with Genoa, with only small bursts of chanting from pockets of fans in the ground coming after Tijjani Reijnders smashed in Milan's third in the 74th minute.
Christian Pulisic's first Serie A double, Reijnders' strike and further goals from Ismael Bennacer and Rafael Leao made it a heavy win which snapped a six-match winless run in all competitions for second-placed Milan, who also reached the quarter-finals of the Europa League this season.
But fans are unhappy about the 18 points which separate Milan from champions and local rivals Inter Milan, who secured their 20th league title by winning their local derby last month.
That, and being dumped out of the Europa League by fellow Italians Roma, angered supporters and means Stefano Pioli, the man who guided Milan to the 2022 Serie A title, will be dismissed at the end of the season after nearly five years at the helm.
