Plzen beat defending champions Atletico Madrid to top their group and are now heavy favourites to progress past the high-flying Italians after late goals from Stanislav Tecl and Frantisek Rajtoral built on Vladimar Darida's first-half opener.
Napoli boss Walter Mazzarri had selected a strong starting XI with competition's top-scorer Edinson Cavani included while new loanee Rolando made his debut. Marek Hamsik was, however, rested on the bench.
Serie A's second-placed side were so subdued in the opening period, however, that Hamsik was sent on at the start of a second period he dominated only to see his side's hopes go up in smoke.
Napoli had begun brightly enough with Goran Pandev's 18-yard shot tipped over by goalkeeper Matus Kozacik.
But from there Plzen, who had not played competitively since beating Atletico on December 6, showed why they had won their group.
Morgan De Sanctis had already needed to tip away a Radim Reznik effort before he was picking the ball out of his net after 28 minutes.
Darida gathered the ball on the edge of the area before blasting a rising shot into the top corner.
A lifeless Napoli could only end the half with a Cavani header that spooned over.
Mazzarri responded by sending on Hamsik for defender Alessandro Gamberini in an attacking move that initially helped spur his side.
Hamsik immediately forced Kozacik into action before a Cavani shot narrowly went wide after the Slovak's assist.
Kozacik did well to keep out Blerim Dzemaili's fierce drive and the goalkeeper had to be quick off his line to block Hamsik after his jinking run left the away defenders in his wake.
It was not all one-way traffic and Rajtoral had a goal ruled out for a foul in the build-up.
Hamsik appeared certain to equalise, smashing a shot narrowly over, before Plzen grabbed a shock second after Camilo Zuniga completely missed his attempted clearance.
That allowed Jan Kovarik to deliver a cross for Rajtoral to force in at the back post.
It got worse for Napoli a minute from the end as another defensive blunder allowed Tecl to spring the offside trap and almost certainly settle the tie.