The Northern Irishman was seen raising a finger on his left hand as he walked off the 11th green at Bethpage Black during his afternoon fourballs match.
He and his partner Shane Lowry had just gone two up against Sam Burns and Patrick Cantlay when he appeared to respond to something in the crowd.
It is not known what provoked the gesture.
On the crowd's antics, McIlroy said: "It was what we sort of expected. It was rowdy and lively. They made it difficult for us.
"You know, but I felt like Shane and I, we handled that really well and dug in. Made some good swings and good putts when we needed to."
Earlier, European Robert MacIntyre had a spat with an American member of the crowd following a comment made about his weight on the 17th tee.
Much had been made of the Bethpage Black crowd in the build-up to the tournament and how they were expected to make it a hostile "bear pit".
But McIlroy had helped keep them largely quiet as he enjoyed an excellent opening morning of the 45th Ryder Cup as Europe were in a strong position.
He continued his winning record with Tommy Fleetwood in the morning foursomes, celebrating a commanding 5&4 victory over Collin Morikawa and Harris English, which helped give Europe a 3-1 lead.
Partnering his friend Lowry, the Irish pair were in a strong position at 2 up after 11 but were pegged back to all square heading into the final few holes.
Captain Luke Donald was not aware of the incident on the 11th, but praised the way his team handled the spectators.
"I'm not aware of that," he said. "But you know, I was definitely proud of the guys, how they handled the situation, a tough environment out there."
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