The defender was carrying the ball forward when he was caught by a lunging slide tackle from behind by Fernando Nino, forcing the Frenchman off injured on the hour-mark at the King Power Stadium.
Paramedics and Leicester's physios immediately rushed on to tend to Fofana before carrying him off on a stretcher and Brendan Rodgers was visibly frustrated with both the Villarreal dugout and players on the pitch.
The details of his injury remain unclear but it potentially leaves Leicester, who are already without the injured Jonny Evans, another defender short just 10 days before the Premier League campaign begins.
Having joined from Saint-Etienne in September 2020, Fofana impressed in his debut season as he made 28 appearances for Rodgers' men, who missed out on Champions League football at the final hurdle.
Despite missing 10 top-flight fixtures, Fofana ranked third for the Foxes in terms of duels won (183), second for aerial success (91) and joint-first for interceptions with Wilfried Ndidi (61 each).
Leicester was leading 3-0 at the time, though only finished as a 3-2 winner over Villarreal, with the La Liga outfit pulling back two late goals.
Çağs opening the scoring ⚽️— Leicester City (@LCFC) August 4, 2021
Thoughts on the first half, #lcfc fans? 🤔
The Foxes, who won the FA Cup against Chelsea last term, have now completed their final pre-season game and next face Manchester City in the Community Shield on Sunday (AEST).