In the other group, qualification will go down to the final day of matches, after bottom side Al Ahly finally recorded a win - beating leaders Wydad Casablanca 1-0.
Zambia's Zesco are tied with Wydad after being held 1-1 by Asec Mimosas of Ivory Coast, who have the same number of points as Ahly.
The loss led to the resignation of Zamalek coach Mohamed Helmi, while captain Hazem Emam has been suspended over an altercation with a team member.
Zamalek chairman accepted Helmi's resignation and has appointed Mohamed Salah  as interim coach.
An own-goal from Zamalek's Ali Gabr in the 79th minute secured Sundowns' qualification to complete a remarkable turnaround for the South Africans in the Champions League.
Sundowns had been eliminated in the final qualifying round for the Champions League last April by AS Vita Club from the Democratic Republic of Congo.
Demoted to the second-tier Confederation Cup, Sundowns were beaten by Medeama of Ghana in a play-off to suffer further African disappointment.
But on the eve of the June draw for the Champions League groups, the organisers kicked out Vita Club for using a banned player and Sundowns were reinstated.
The victory over Zamalek puts Sundowns top of Group B on nine points - six ahead of Zamalek in second.
We still have to do the business
"The gap is getting closer and closer between us and the north Africans," Sundowns coach Pitso Mosimane said after the win.
But he cautioned that Sundowns' hard work could come to nothing if they fail to win the title.
"There's a little bit of bragging rights in beating Zamalek, but we still have to do the business," he said.
Bottom side Enyimba of Nigeria are yet to register a point after two matches in this group which is reduced to three teams following Entente Setif's disqualification.
Enyimba and Zamalek play in the final round of group matches to decide which of them will join Sundowns in the last four.
In Group A, Wydad Casablanca of Morocco and Zesco United of Zambia also had the chance to secure semi-finals places if they both won their matches on Wednesday.
However, Zesco United were held to a draw away to Asec Mimosas in Abidjan to open up the group and keep ASEC's faint hopes of progressing alive.
Asec piled on the pressure on Zesco with a 75th minute strike from Nabi Kone, but the visitors responded five minutes later with an equaliser from Jackson Mwanza.
The result left Zesco level on seven points with leaders  Wydad Casablanca who were beaten at home by bottom-of-the-table Al Ahly of Egypt in the late match.
Asec are a further three points behind.
Ahly had managed just one point from their first three games but Rami Rabia's goal early in the second half in Morocco gave them a fine away win.
Ahly play Zesco and Asec take on Wydad in the final round of matches, so all four teams from Group A still have hopes of going through to the last four.
Mamelodi Sundowns of South Africa continued their impressive run in the African Champions League with a 1-0 win over Zamalek on Wednesday which put them into the semi-finals.