The defending champions came from behind against their Egyptian rivals to progress in an exciting encounter at El Gouna Stadium.
Omar Gaber put Zamalek in front after just four minutes, but Waleed Soliman quickly equalised for the hosts with his third goal of the competition.
Striker Ahmed Abdul Zaher then gave Al Ahly the lead before Mohamed Aboutrika and Ahmed Fathy put the game beyond doubt in the second half, Soliman providing the assists for both.
Zamalek - who have won the tournament five times in their history - did pull one back through substitute Ahmed Hassan, but by that point their elimination from the competition had been all but confirmed.
Al Ahly sit top of Group A, their place in the last four sealed by virtue of their head-to-head record against Leopards.
Cameroonian side Cotonsport, who reached the final in 2008, will also be in the next round following their goalless draw with Ivorian outfit Sewe Sport.
Sewe headed into the game knowing they needed to take all three points to have any chance of progressing, but were unable to break through in front of their home fans at the Stade Robert Champroux.
That result renders the final two games in Group B meaningless after ES Tunis secured their progression on Saturday.
The final group fixtures are set to take place on September 21 and 22 with Orlando Pirates and Leopards, who face Al Ahly and Zamalek respectively, out to try and clinch the last semi-final spot.
