The Germans needed a victory to keep their qualification hopes alive, and they got one thanks to goals from Marco Reus, Jakub Blaszczykowski and Pierre-Emerick Aubameyang.
Last season's runners-up dominated large parts of the Group F encounter and went ahead through an early Marco Reus penalty, after Robert Lewandowski had been fouled.
Jakub Blaszczykowski doubled the advantage on the hour mark and Pierre-Emerick Aubameyang netted a third after Lorenzo Insigne had pulled one back for Napoli.
The win - Dortmund's first in four matches following three successive defeats in all competitions - lifts the Bundesliga side to second in Group F, level on points with third-placed Napoli with one match left to play.
Dortmund made one change to the side that lost 3-0 at home to Bayern Munich on Saturday, with Sebastian Kehl coming in for the ineligible Manuel Friedrich.
Napoli, which, like its hosts, had lost its last two domestic fixtures, dropped Miguel Britos, Gokhan Inler and Insigne in favor of Federico Fernandez, Blerim Dzemaili and Dries Mertens.
The match got off to the best possible start for Dortmund, which was awarded a 10th-minute penalty when Fernandez was penalized for holding on to Lewandowski in the area.
Reus made no mistake from the spot, slotting into the bottom right-hand corner as Pepe Reina went in the opposite direction.
Lewandowski should have doubled the advantage five minutes later when he guided the ball wide of the target from just inside the 18-yard box, having been played in superbly by Henrikh Mkhitaryan.
Reina got down well to his right to deny a Reus free kick from 20 yards moments later as Jurgen Klopp's men continued to pile on the pressure.
Napoli began to find its feet as the half progressed with Goran Pandev sending its first clear-cut chance straight at Dortmund goalkeeper Roman Weidenfeller.
Jose Callejon went even closer for the visitors on the half-hour mark but was denied by the left upright after driving an effort across the face of goal.
Dortmund, though, refused to sit back and defend its lead and Reina had to be on top form to keep out strikes from Mkhitaryan and Lewandowski before the break.
That pressure, which continued after the interval, paid off in the 60th minute as Dortmund broke following a saved Gonzalo Higuain effort. Reus found Blaszczykowski in space 18 yards out and the unmarked midfielder slipped the ball under an advancing Reina.
However, Napoli put itself right back in the game in the 71st minute as Insigne charged forward and cut a low left-footed strike into the back of the net off the right-hand post.
Aubameyang restored Dortmund's two-goal advantage seven minutes later, though, chipping over Reina after receiving the ball from Lewandowski.
Dortmund's victory means a win over Olympique de Marseille on matchday six will see it progress, while Napoli must defeat Arsenal at the San Paolo to keep its hopes alive.