Two first-half headers by Mohamed Fadl put Ismaili in the driving seat to book a last-four berth.
Esperance needed to find the net to level the aggregate score, having defeated Ismaili 2-1 at home, and their hope was fulfilled when some sloppy defending gifted Michael Eneramo a 50th-minute goal.
Neither side could notch the crucial goal during the remaining minutes.
The ACL regulations do not include extra-time as the match went directly to penalty kicks.
Fadl's opening spot-kick was blocked by Esperance keeper Hamdi Kasraoui and Abdullah Saied blasted Ismaili's fifth effort high over the bar.
Esperance converted their kicks to oust Ismaili, who will have to cause an upset in the Egyptian league and dethrone leaders Ahli if they are to avoid ending the season empty handed.
Ismaili trail Ahli, who aim to win a fifth consecutive league title, by four points with eight matches to go.
Small Crowd
Ismaili had to play in front of only 9,100 of their supporters as their stadium is undergoing renovations ahead of the U-20 World Cup that Egypt will host in September.
Esperance almost stunned that crowd after six minutes when Wajdi Bouazzi crossed low for Eneramo, who shrugged off Dario Khan but his subsequent shot missed the target from four yards out.
Ismaili then made sure Esperance would be busy thwarting their attacking attempts rather than trying to score a precious away goal.
The Dervishes were largely helped by the energetic moves of wingers Ahmed Samir Farag and Omar Gamal.
Gamal's in-swinging corner after 26 minutes found Fadl, who towered above his marker to head home and give Ismaili the lead.
Farag was unlucky to see his fierce low drive strike the woodwork 11 minutes later.
The hosts' pressure paid dividends once again as Ismaili went further ahead five minutes before the break.
Fadl superbly headed Farag's pinpoint cross from the left past the hapless Kasraoui despite the latter's desperate attempt to keep the ball out.
Defensive Lapse
Although Ismaili were in firm control during the first-half, a defensive lapse in the second cost them a clean sheet that could have lifted them through to the semis.
Five minutes after the restart, Oussama Darragi skipped past two defenders and jinked his way into the area.
He sent in a low cross for Eneramo who held off his marker far too easily to slot home from close range.
Ismaili dominated possession afterwards but barely posed a threat as Esperance had the upper hand in the penalty shootout to go through.