Left-back Leonel Vangioni suffered a horrendous moment in first-half injury time, getting caught on the ball as Estudiantes' Diego Vera stole possession to score the opener past a stranded Marcelo Barovero in the River Plate goal.
Vangioni was substituted six minutes into the second half to cap a poor night personally in Buenos Aires but Marcelo Gallardo's men fought back to take a lead into their second leg at home on November 6.
Rodrigo Mora equalised in the 53rd minute, nodding home from a Carlos Sanchez cross from the right.
River Plate got a valuable second goal with some pluck, Sanchez's attempted cross in the 71st minute was deflected into the net off the head of Estudiantes' Jonathan Schunke - who was credited with the own goal.
Alejandro Bernal struck the winner for Atletico Nacional, as they took a 1-0 lead after the first leg of their last-eight tie with Cesar Vallejo.
Bernal's deflected strike from range found the back of the net in the 57th minute, to hand the Colombians the advantage ahead of the return leg in Peru.