The Strongest suffered a last-gasp 2-1 defeat at Trujillanos as their qualification hopes for the Copa Libertadores knockout rounds were left up in the air.
With the game petering out to a draw, Trujillanos midfielder Maurice Cova converted a 94th-minute penalty to lift the Venezuelan hosts to their first win in Group 1 on Tuesday.
The Strongest, who saw defender Federico Pereyra sent off in the 89th minute for a second bookable offence, thought they had earned a share of the spoils after Matias Alonso's 67th-minute effort cancelled out Carlos Sosa's first-half opener.
Back-to-back defeats left The Strongest in second position, two points ahead of Sao Paulo, who are scheduled to play leaders River Plate on Wednesday, and one adrift of the Argentine giants with one match remaining.
Already assured of a place in the round of 16, Atletico Nacional preserved their 100 per cent record thanks to a 1-0 win against Sporting Cristal.
Victor Ibarbo, on loan from Cagliari having spent time at Roma and Watford, scored from the penalty spot in the 13th minute.
Ibarbo's penalty gave Nacional their fifth win from as many games without conceding a goal in Group 4.
Second-placed Huracan were also in action on Tuesday, playing out a goalless draw at home to winless Penarol.
Meanwhile, Group 6 leaders Toluca remain unbeaten after Fernando Uribe scored twice inside six minutes late in the second half to inspire a 2-1 victory over San Lorenzo.