Michal Sadilek’s early opener had the Czechs on course for just their second World Cup win as an independent nation.
But Mokoena’s late spot-kick kept both teams in the hunt for the last 32, although they will almost certainly have to win their final Group A fixture to advance.
Both sides move onto one point, two behind co-hosts Mexico and South Korea, who face off later on Thursday.
South Africa next face South Korea, while the Czech Republic take on the daunting task of playing Mexico in the Estadio Azteca.
Both sides started with disappointing performances and defeats on the World Cup’s opening day, but the Czechs quickly set about making amends.
Patrik Schick is the biggest name in a Czech side lacking the star quality of previous generations but the Bayer Leverkusen forward missed a glorious chance inside the first minute when he planted a header wide.
Miroslav Koubek’s men only had to wait another five minutes to take the lead.
Adam Hlozek’s cross was classily cushioned by Alexandr Sojka into the path of Sadilek, who calmly stroked the ball past Ronwen Williams.
Now in their fourth attempt, South Africa have never progressed beyond the group stage at the World Cup.
Hugo Broos has overseen an upturn in Bafana Bafana’s fortunes since taking charge five years ago.
The 2010 hosts had not qualified for the World Cup since 2002 but finished third at the 2023 Africa Cup of Nations.
Their return to the global stage has exposed a lack of quality, though, particularly in forward areas.
The Czechs were left to rue not making more of their chances to kill the game off early in the second half.
Vladimir Darida took too long to get his shot off with a big chance before Lukas Cerv’s long-range effort was tipped over by Williams.
There were thousands of empty seats on show at the futuristic home of the NFL’s Atlanta Falcons, but those that were in attendance loudly booed the mid-half hydration breaks in an enclosed and air-conditioned stadium.
The majority of the crowd were backing South Africa and finally had something to cheer seven minutes from time.
Thapelo Maseko’s shot hit the arm of the unfortunate Pavel Sulc and referee Tori Penso, part of an all-female officiating team, pointed to the spot.
Mokoena confidently drilled home the spot-kick for his nation’s first World Cup goal in 16 years.
They came close to a second five minutes later when Matej Kovar saved from Relebohile Mofokeng.
But both sides had to settle for a point that could yet prove vital come the end of Wednesday’s final Group A games.
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