The Argentinian side were beaten 2-1 by the Ecuadorian outfit last week but got a valuable away goal in Guayaquil.
Match preview
Estudiantes face elimination from the Copa Sudamericana if they cannot reverse the loss suffered in Ecuador against Barcelona.
A crucial away goal scored by Leonardo Godoy brought Eduardo Dominguez's level, but El Leon could not hold on for long before conceding a 78th-minute goal.
Overturning the loss is not impossible, although a poor run does not bode well for the home side, whose two wins in 11 games in all competitions gives little reason for optimism.
Five previous winless games, including the 2-1 loss on July 12, suggest a victory might be a tall order for the Argentinian club.
Estudiantes have netted more than one goal in one of their previous nine games leading up to Wednesday — a 4-0 triumph in their final Sudamericana group game against Oriente Petrolero — further underscoring the task faced by the home side.
Those statistics should be music to Barcelona's ears, given their slightly better form than Wednesday's opponents.
Diego Lopez was appointed recently, with this midweek's encounter the manager's first in the dugout since taking charge, and the Uruguayan seeks a positive start.
Unlike Estudiantes, Los Canarios did not start their continental journey this season in the Sudamericana, instead exiting the Copa Libertadores after the group stage.
The Ecuadorian side ended a distant third behind Palmeiras and Bolivar, picking up one victory from six games.
They finished without a point on their travels, losing each game with seven goals conceded in three matches, averaging just over two goals against in three road defeats.
Estudiantes Copa Sudamericana form:
W
D
W
D
W
L
Estudiantes form (all competitions):
W
D
D
D
L
L
Barcelona SC Copa Sudamericana form:
W
Barcelona SC form (all competitions):
W
L
L
L
D
W
Team News
Pablo Piatti remains sidelined after rupturing cruciate ligaments since April, with the 34-year-old ruled out for the season.
Mariano Andujar was not in the Estudiantes squad against River Plate on Sunday, but the experienced goalkeeper should return for Wednesday morning's clash.
Dominguez is not expected to veer away from utilising a back three, usually a 3-5-1-1.
Luis Arce is a long-term absentee with a knee injury for Los Canarios, ruling out the midfielder for Wednesday.
The Ecuadorian side tends to utilise a 4-2-3-1, but it is anyone's guess as to how Lopez approaches the round of 32 tie.
Estudiantes possible starting lineup:
Andujar; Mancuso, Lollo, Nunez; Godoy, Zuqui, Rodriguez, Ascacíbar, Benedetti; Rollheiser; Carrillo
Barcelona SC possible starting lineup:
: Burrai; Perlaza, Rodriguez, Sosa, Portocarrero; Gaibor, Almeida; Martinez, Diaz, Corozo; Fydriszewski
We say: Estudiantes 2-0 Barcelona SC (Estudiantes win 3-2 on aggregate)
Despite their indifferent results, Estudiantes' impressive home form — unbeaten since February in all competitions, winning eight and playing out six draws — should carry them to the round of 16 at the expense of Barcelona SC.