The Uruguayan outfit had quickfire goals from Juan Amado and Giorgian De Arrascaeta early in the second half to help them to victory at the Estadio Huancayo in Peru.
Defensor have 10 points from five games, and are all but assured of their spot in the knockout stages, although Cruzeiro and Universidad Chile have a game in hand.
Matias Alonso was the creator of Defensor's first goal on 50 minutes, as his dinked through-ball release Amado into acres of space.
It was Amado's first goal for his club since May 2012.
Defensor then caught Garcilaso on the counter to double their lead four minutes later, with Felipe Gedoz drawing Real's two central defenders to play in De Arrascaeta, who chipped goalkeeper Diego Carranza to score.
Defensor finish their group stage fixtures at home to Universidad Chile on April 9, while Real Garcilaso are on the road at Brazil's Cruzeiro.
