Paraguay's Cerro Porteno also reached the last four with a 1-0 home win over Chiapas of Mexico in the second leg of their quarter-final tie to qualify 2-1 on aggregate.
Penarol, who last reached the semi-finals in 1987 when they won their fifth title, lost 2-1 away to Chile's Universidad Catolica in Santiago but advanced 3-2 on aggregate.
Catolica took the lead in the 18th minute when striker Lucas Pratto shook off a challenge and laid the ball off to midfielder Fernando Meneses, who raced through to fire into the net.
Substitute Roberto Gutierrez levelled the aggregate score in the 69th minute when he turned central defender Guillermo Rodriguez and unleashed a low shot into the bottom corner.
As the match looked set for a penalty shootout, with no extra-time in the competition, former Uruguay midfielder Estoyanoff ghosted in on the right to tap in a cross in the 85th minute.
"We were the better team but we made two mistakes over there (in Montevideo) that lost us the tie," Argentine Pratto, Catolica's top scorer with six goals, told Fox Sports.
"This win is doubly gratifying because Catolica are a great team, they play really good football," said Penarol striker Juan Manuel Olivera.
Penarol advanced to meet Velez Sarsfield of Argentina in the semi-finals.
Velez, who completed a 7-2 aggregate victory over Libertad of Paraguay on Wednesday, will be at home in the second leg as they aim to claim a second title after previously winning the tournament in 1994.
In Asuncion, central defender Pedro Benitez headed the only goal 20 minutes from time following a poor clearance at a corner to put Cerro Porteno in the semi-finals for first time since 1999.
Chiapas' Chilean international defender Ismael Fuentes was sent off eight minutes from time for his second booking in six minutes.
In the semi-finals, Cerro face twice champions Santos of Brazil, who drew 1-1 at home to Colombia's Once Caldas on Wednesday to go through 2-1 on aggregate.
