Although the first leg had ended in a 2-1 away win for Barcelona SC, a three-goal deficit was imposed by the continent's soccer authority Conmebol as a punishment for the Ecuadorian club playing midfielder Sebastian Perez, who was only allegedly ineligible as his registration was only finalised after the Copa Libertadores deadline.
Barcelona SC have said they will appeal the ruling, blaming the Ecuadorian Football Federation for the delay in processing the player's transfer documents.
A Conmebol committee agreed with Defensor's appeal, and awarded the Uruguayan side the three points and a three goal lead.
With that cushion, Defensor sat back and controlled the game, remaining organised defensively and stopping Barcelona's advances down the wings.
The Ecuadorians were up against it, and they allowed nerves and tension to creep into their game, as they failed to create any clear chances and were hampered by tame or wayward finishing.
Matias Oyola, in for the controversially missing Perez, scored the game's only goal shortly before half time, calmly gathering a loose ball that had been only partially cleared by the Defensor goalkeeper before lifting a curling effort into the empty net from outside the penalty area.
Barcelona SC pressed forward in the second half, but could not break through Defensor's impressive rearguard.
As time trickled away and the missed chances piled up, hope slowly drained from the home team and its fans, and Barcelona SC slipped tamely out of the competition, although the manner of their elimination after Conmebol's ruling will be difficult to swallow, after their first leg victory away in Montevideo was overruled by a tribunal.
In the meantime, however, the Uruguayans advance to the third round, where they will face Brazil's Atletico Mineiro, who beat Danubio.