The third quarter belonged to the Madibaz, who stunned the champions as goal shooters Bianca Blom and Nolusindiso Twani flourished to put them 28-26 ahead.
In a tense final quarter, the underdogs delivered a classy performance, putting up a brilliant fight against the favourites to clinch an unexpected victory.
Earlier, Pukke goal shooters Marna Claassens and Fifi Tsotetsi showed their accuracy in the circle to give their side a 12-7 lead in the first quarter, extending that to 20-14 in the second quarter.
The Madibaz, however, showed character against a strong Pukke team to come back strongly, moving to fourth spot on the log.
It was the first time since 2005 that Maties had defeated Kovsies, knocking the two-time champions out of the play-offs.
The score was close in the first two quarters, with Maties leading 24-21 at the second break.
The defining passage came in the final quarter when the Maties, who had led 39-30 at the end of the third quarter, exploded into life to score 15 goals to the eight of Kovsies.