Penarol stunned the home crowd at the Estadio Nuevo Corona when they hit the front in the opening minute but the visitors were unable to stop Santos Laguna in the second half, as the home side struck three times after the break to register a comfortable victory.
The win was Santos' fourth in Group 8 with the Mexican club staying unbeaten in five matches in the continental competition.
Penarol took advantage of a defensive mix-up just after the opening kick-off when Javier Toledo nodded the ball past Santos goalkeeper Oswaldo Sanchez.
But the home team were level just eight minutes later thanks to Carlos Quintero's volley from Jonathan Lacerda's lofted diagonal pass.
Quintero was crucial to Santos' victory with the Colombian winger creating his team's second goal just before the hour-mark, picking out Javier Orozco, who burst in behind Penarol's defence to score.
It was 3-1 in the 78th minute when Quintero lifted the ball over Penarol goalkeeper Juan Guillermo Castillo.
The Colombian capped off a brilliant individual performance with an assist for Santos' fourth goal, playing a pass in behind to Andres Renteria, who converted from a tight angle.
The win took Santos (13 points) seven points clear at the top of Group 8 with Argentina's Arsenal in second, while Penarol (four points) are two ahead of Deportivo Anzoategui of Venezuela.
In Group 4, Zamora trumped Nacional Asuncion 2-0 with second-half goals from Juan Falcon and John Murillo.
The Venezuelan hosts hit Nacional twice on the break after half-time with Pedro Ramirez creating both goals.
The win took Zamora to seven points from five games, while Paraguay's Nacional have five.
Reigning Copa Libertadores champions Atletico Mineiro remain top of the group with eight points from four matches.
