The Bolivian hosts scored twice within 10 minutes late in the second half in La Paz, to build a comfortable lead ahead of the return leg in Uruguay on April 29.
A scoreless first half saw Defensor confident of heading to their Montevideo home full of confidence, but The Strongest broke the deadlock on 69 minutes.
Pablo Escobar's chipped ball into the area was on the head of Jair Reinoso, who buried the header despite Defensor goalkeeper Martin Campana getting a hand on the shot.
Ten minutes later, Raul Castro's long-range effort bounced over Campana at his near post, much to the delight of the home faithful.
Eduardo Villegas' men are on track to continue their club-record performance in the continental competition, with their previous best efforts seeing them eliminated in the group stages.
Defensor, meanwhile, will need at least two goals in their home leg, if they are to match their last-eight efforts in 2007 and 2009.
