Jeremy Brockie was benched by Eric Tinkler, affording the Kiwi striker a chance to catch his breath at the end of a tiring month. Meanwhile, Bradley Grobler started for Matsatsantsa.
The game got off to a slow start, with SuperSport showing more promise than Ajax going forward, but neither side truly troubling the opposition keeper early on.
That changed in the 25th minute, when Richard Boateng picked out Aubrey Modiba with a cross from the left and he headed into the bottom corner for the opener.
Ajax were lacking incisiveness throughout the first half and Stanley Menzo responded, making an early substitution in the 41st minute as he brought Sedwyn George on for Masilake Phohlongo.
However, two minutes later, SuperSport nearly doubled their lead. It took an alert save from Jody February to keep out Grobler's low shot.
Ajax immediately looked more promising at the start of the second half, with captain Mosa Lebusa regularly bombing forward and causing problems for the Matsatsantsa defence.
Tashreeq Morris forced a fine save from Reyaad Pieterse with a powerful strike in the 58th minute.
Perhaps alarmed by the way the complexion of the game had changed, Tinkler brought Sipho Mbule on for Fagrie Lakay in the 60th minute.
Eleven minutes later, Menzo brought Yagan Sasman on for Rheece Evans before unleashing a tirade at the former Sundowns man as he made his way off.
There was a controversial moment in the 74th minute. Referee Eketsang Setloboko dropped the ball, seemingly for Pieterse, but Ajax put it in the back of the net. The goal was disallowed, much to the disappointment of the Urban Warriors.
Five minutes later, Brockie came on in place of Grobler for a late cameo appearance. Menzo then made his final change in the 85th minute as Toriq Losper replaced Bantu Mzwakali.
The last substitution came in the 91st minute, when Tefu Mashamaite replaced Reneilwe Letsholonyane.
Despite Ajax's spirited second-half effort, Matsatsantsa held on for the win. They now trail leaders Baroka by a mere six points with six games in hand, putting them very much in the mix in the Premiership title race.