Ibrahimovic has been out of action since his team's Serie A opener against Lazio earlier this month with an ankle injury.
The Sweden international also missed his team's 2-2 result at Barcelona in their Group H opener.
Ambrosini dislocated his shoulder in that game but could also feature on Wednesday.
"We are evaluating Ibrahimovic and Ambrosini's condition," Milan coach Massimiliano Allegri said."They are definitely better and we hope they will be ready for Wednesday's game."
The Rossoneri go into the encounter at the San Siro on the back of Saturday's 1-0 triumph over Cesena.
Clarence Seedorf's goal handed the Italians their first win in Serie A this season.
"It's a delicate moment for us considering the number of players that are injured," Seedorf said. "But we are proving that we are a team.
"This was very important because it was our first and now we can fully focus on Wednesday's game against Plzen."
Milan were disappointingly knocked-out of the Champions League by Tottenham at the last-16 stage last year and hope to go all the way this season.
"Last year we struggled in the Champions League," Seedorf said. "But this season we want to aspire to the maximum in this competition."
Milan are still without forwards Alexandre Pato, Robinho and Kevin-Prince Boateng, as well as midfielders Gennaro Gattuso and Mathieu Flamini and defenders Luca Antonini, Philippe Mexes and Daniele Bonera.
Tournament newcomers Viktoria visit Italy for the first time looking to keep up their great European form on the road this season.
The Czech champions won three successive away games en route to the group stage.
Pavel Vrba's men, who drew 1-1 at home to BATE Borisov in their group opener, lie fourth in the Czech first league.
"We want our opponents in the group to feel we can seriously do something and are not here just to get beaten in every game," Vrba said.
Plzen were held to a goalless result at Ceske Budejovice last Friday.