Chiefs were two goals down by halftime before launching a spirited comeback, but they were unable to force an equaliser that would have taken the match to extra time.
Four-time winners Mamelodi Sundowns edged through after a difficult encounter against second-tier Gomora United, securing a 2-1 victory.
Stellenbosch took the lead in Cape Town from the penalty spot when Nigerian midfielder Ibraheem Jabaar converted after 10 minutes.
The hosts doubled their advantage with a superb strike from Devin Titus, who collected the ball 30 yards from goal and rifled his effort into the back of the net.
Chiefs forced several excellent saves from Stellenbosch goalkeeper Sage Stephens and also struck the woodwork on a couple of occasions on an unlucky night for the 2025 champions.
They reduced the deficit when forward Flávio Silva converted from the penalty spot in the 56th minute, but were unable to find a second goal.
Both sides had further chances to score thereafter, but there were no additional goals.
The result provided revenge for Stellenbosch, who were beaten 3-1 by Chiefs last season in the Nedbank Cup as AmaKhosi went on to lift the trophy.
Sundowns were made to work hard for their win as Gomora put up a spirited display in their Nedbank Cup debut. The Brazilians extended their record of never having lost to lower-league opposition in the competition, a run that now stands at 24 matches (W23 D1).
Peter Shalulile scored the opener to extend his record as the leading scorer in Nedbank Cup matches to 15 goals, moving further clear of Hlompho Kekana and Kermit Erasmus, who have each scored 11.
Mpho Maoke netted his first-ever goal in professional football to equalise for Gomora just before halftime, sending the teams into the break level at 1-1.
However, Sundowns’ greater experience told in the second half, and Thapelo Morena, who has a rich history in the competition after scoring the winning goal in the 2022 final, struck just past the hour mark to seal a 2-1 victory.
