The Brazil international was on the scoresheet during the league fixture, finding the back of the net in the 73rd minute to make it 2-0 to Los Blancos, with Federico Valverde breaking the deadlock in the first period.
Vinicius had to be replaced in the 79th minute, though, and Ancelotti has revealed that the forward will need to undergo an examination before the seriousness of the damage is confirmed.
"He's had a cervical problem and is completely blocked. He's in pain and will be tested in the next few hours," Ancelotti told reporters during his post-match press conference.
A cervical issue suggests that he has pain in his neck, but it has also been reported that the 24-year-old has an issue with his shoulder, which could see him miss out for Brazil over the international break.
Vinicius suffers "cervical problem" against Villarreal
Vinicius has again been in strong form for Real Madrid during the 2024-25 campaign, scoring four goals and registering seven assists in 12 appearances in all competitions.
Los Blancos are not back in action until October 19 against Celta Vigo, and they will then tackle Borussia Dortmund in the Champions League and Barcelona in Spain's top flight in the latter stages of the month.
Real Madrid will be missing Dani Carvajal for the remainder of the season after the defender suffered a severe knee injury during the league fixture with Villarreal.
Carvajal picked up the issue in the latter stages of the contest at Bernabeu, and the Spain international will not be back on the field until the 2025-26 campaign.
Ancelotti pleased with Real Madrid "attitude" against Villarreal
Real Madrid entered the clash with Villarreal off the back of a 1-0 loss to Lille in the Champions League, but they were able to return to winning ways in Spain's top flight.
"We played with discipline and a good attitude. Without too much intensity, but when we broke the deadlock with a rehearsed move we controlled well, without taking risks and defending solidly. It could have been a tricky game against a team that is in good form and we picked up three important points," said Ancelotti.
"We rehearsed Valverde's goal this morning and the move was to free Federico on the edge of the box. Today we scored two goals from the edge of the area and they're assets in the squad because we have players with great shots like Vini Jr, [Kylian] Mbappe, Valverde, [Jude] Bellingham. It worked out well."
Real Madrid's unbeaten run in La Liga has now been stretched to 41 matches, with the capital giants last losing in Spain's top flight to Atletico Madrid in September 2023.