The visitors eased through the group stage, dropping just two points, while the Austrian side dropped out of the Champions League, finishing third in their group, resulting in a place in the final 32 of the Europa League.
The home side were fighting an uphill battle in the Champions League, having been drawn in a group with Bayern Munich and Atletico Madrid, however they managed to secure the third spot with a 3-1 win over Lokomotiv Moscow, allowing them to compete in the Europa League.
Jesse Marsch's side have remained dominant domestically this season, currently on track for their seventh consecutive Austrian title.
Die Roten Bullen sit three points ahead of second-placed Rapid Vienna, having won 13 of their 17 matches in league action.
That is largely down to their strong attacking record, with no side getting close to their 54 goals from 17 games.
Meanwhile, only two teams in the Austrian top flight have conceded fewer than the 18 they have shipped, showing that Marsch has built a balanced and effective side.
They held their spot at the summit of the table with a dramatic win last time out, when they came back from being 2-1 down with 10 minutes to go.
Mergim Berisha equalised for Salzburg, before Patson Daka netted his second of the game to put Marsch's side ahead, with Sekou Koita doubling the lead in added time to see the side from the Austrian capital record a 4-2 victory.
That result stretches Die Roten Bullen's winning run to six matches in all competitions, and they will be hoping to extend that to seven with a victory in this first-leg clash on Wednesday.
Villarreal topped their Europa League group with 16 points, having scored 17 goals from their six matches.
However, Unai Emery's side have hit a tougher patch of form in recent weeks, having only won one of their last seven, and remaining five games without a victory in La Liga.
Most recently, the Yellow Submarine fell to a 2-1 defeat at home to Real Betis, with Gerard Moreno scoring a penalty in the second half, after the visitors led 2-0.
As a result of their disappointing form, Villarreal have dropped from third to sixth in La Liga, having previously been on track for Champions League qualification.
That means Emery will be desperate to go deep into this season's Europa League, as winning the trophy could be their only path into continental football next season.
The Spaniard is one of the most qualified men to bring home the trophy too, having guided Sevilla to three consecutive Europa League titles between 2014 and 2016.
Red Bull Salzburg form (all competitions): WWWWWW
Villarreal Europa League form: WWWDWW
Villarreal form (all competitions): DWDLDL
The home side remain without centre-back Maximilian Wober, who is carrying a thigh injury.
Since the departure of Dominik Szoboszlai to RB Leipzig in January, Jesse Marsch's attack has been led by Patson Daka, who has registered 13 goals and five assists in 13 league appearances this season.
Villarreal have no shortage of injuries to deal with for this match, with experienced midfielders Vicente Iborra and Francois Coquelin sidelined, while January signing Etienne Capoue was only fit enough to make the bench on Sunday.
Their attack has also taken a hit, with Samu Chukwueze out with a groin injury.
Emery's attack will likely be led by the exciting combination of Gerard Moreno and Paco Alcacer, who have netted a combined 22 goals in all competitions this season.
The final attacking spot could be up for contention, with Moi Gomez and Fernando Nino both laying their claims to starting places.
Red Bull Salzburg possible starting lineup:
Stankovic; Kristensen, Solet, Vallci, Ulmer; Junuzovic, Mwepu; Berisha, Sucic; Daka, Adeyemi
Villarreal possible starting lineup:
Asenjo; Pena, Albiol, Torres, Pedraza; Baena, Foyth, Parejo; Moreno, Alcacer, Gomez
We say: Red Bull Salzburg 2-2 Villarreal
We see this game being a very open and exciting encounter, as two good attacking sides square off at the Red Bull Arena, with neither team coming out on top in this first leg.
While Alcacer and Moreno could have a field day against a weakened Salzburg defence, the attacking threat of Marsch's team, led by the exciting Daka, will cause Villarreal plenty of problems of their own.