Brazilian champions Fluminense lost 2-1 at Ecuador's Emelec after conceding an own goal and a penalty.
Jadson gave Sao Paulo an early lead at the Morumbi with Ronaldinho equalising just before half-time. Diego Tardelli scored the winner in the 60th minute.
It was the third meeting between the Brazilian rivals in the competition and sweet revenge for Atletico, whose perfect record in the group phase was ended when Sao Paulo beat them 2-0 two weeks ago.
In an incident-packed first half, Sao Paulo centre back Lucio was sent off for a dangerous challenge on Bernard, his second booking, leaving his side a man short from the 35th minute on.
"The game was set in a certain way and it took a different turn after (Lucio's dismissal). Unfortunately, any loss of a player is fatal when two balanced teams meet," Sao Paulo captain Rogerio Ceni told Fox Sports.
"We made the most of our qualities, our shooting, our pedigree. It's always like that, we have a different spirit when we play for something," said Ronaldinho.
Ronaldinho had angered Sao Paulo players when he said the teams' previous meeting had been nothing more than a training session for Atletico, who had already qualified.
Sao Paulo began in fiery mood and took the lead in the ninth minute when Paulo Henrique Ganso sidestepped two defenders inside the box and squared to Jadson, who fired a shot past goalkeeper Victor.
The home side were forced to make a substitution immediately afterwards because Aloisio injured his groin making the cross to Ganso and was replaced by Ademilson.
The substitute was guilty of a glaring miss soon after coming on, firing a chance over the bar when he had only Victor to beat.
Atletico equalised three minutes before half-time when Bernard hit a corner well beyond the far post where the unmarked Ronaldinho steered a header back across the face of goal past a static Rogerio Ceni.
Tardelli scored the winner on the hour mark, running on to a through ball from fellow striker Jo to slot past the goalkeeper and give Atletico the advantage for the return leg next Wednesday at the Independencia in Belo Horizonte.
In Guayaquil, Emelec went ahead in the 32nd minute when central defender Leandro Euzebio turned the ball into his own net trying to cut out a cross from right back Carlos Vera.
"Flu" pulled level just before the interval with a superb left-footed shot by midfielder Wagner from outside the box.
With the final whistle just minutes away, Carlinhos brought down Emelec striker Marcos Mondaini and Fernando Gaibor scored the winner with a well-taken penalty out of reach of the diving Diego Cavalieri.
- Sports & Recreation
- Diego Tardelli
- Sao Paulo
- Paulo Henrique Ganso