Penarol, who won the first two of their five crowns in 1960 and 1961, benefited from two goalkeeping blunders by Paulo Garces to beat Universidad Catolica 2-0 at the Centenario in Montevideo.
Santos, whose two titles in South America's elite club competition came in 1962 and 1963, won 1-0 away against 10-man Once Caldas, the Colombian team that had upset favourites Cruzeiro.
Santos went ahead two minutes before half-time when Neymar delivered a perfectly weighted diagonal pass to the unmarked Alan Patrick, replacing injured Brazil playmaker Paulo Henrique Ganso, who shot low past goalkeeper Luis Martinez.
The only Brazilian side left in the competition after four were knocked out in the first knockout round, Santos almost increased their lead in the 65th when Elano's curling free-kick from the left hit the woodwork.
Caldas, who had defender Elkin Calle sent off on the hour for a second booking, were unlucky not to be awarded a penalty in added time when striker Wason Renteria was brought down.
However the Colombian side, who have shown better away form in the competition, will fancy their chances of turning the tie around in the second leg in Santos next week.
KEEPER'S BLUNDERS
At the Centenario, Garces dropped a cross and striker Juan Manuel Olivera tapped the loose ball into the net to put Penarol ahead with his fifth goal in the competition in the 37th minute.
With the final seconds of added-time ticking over, Garces spilt a long punt into his box and lurking Argentine striker Alejandro Martinuccio slotted in to give the Uruguayan side an unexpected two-goal advantage for next week's second leg.
"It's a good advantage but we must go out to play them as equals in Santiago. They are very strong at home," Man of the match Martinuccio told Fox Sports.
"We've already seen in this Libertadores Cup that teams can win at home or away," he added recalling that Penarol upset Brazilian holders Internacional in Porto Alegre in the first knockout round.
Catolica came closest to a goal when defender Dario Rodriguez blocked striker Lucas Pratto's shot on the line.
Two Paraguayan teams are in action on Thursday when Libertad visit Argentina's Velez Sarsfield, champions in 1994, and Cerro Porteno are away to Chiapas in Mexico.
