According to The Sun, Wainwright, 30, was seen touching the knee of the singer — wed to actress Jessica Biel, 37 — before he took hold of her hand.
The pair was snapped around midnight chatting and drinking in a bar in New Orleans while on a break from filming a movie in which they play lovers.
Onlookers were shocked to see him hold hands with Wainwright, and risk upsetting Biel — his wife of seven years and mum to their son Silas, four.
One said: “They were smiling and laughing.
“At one stage he grabbed her hand and rested it on his knee. She then gently started stroking his leg.
“Then he clutched her hand with both of his and was playing with her hands.”
However, a source close to Timberlake dismissed the story in a statement to Page Six.
“He was on an open balcony. They were among friends. It was nothing. There is 100 per cent nothing going on,” the insider said.
Timberlake had been snapped wearing his wedding ring earlier in the week but was not seen with it on during the night out.
The pair, currently filming American football drama film Palmer, were part of a small group out drinking at The Absinthe House, on Bourbon Street, in New Orleans on Thursday.
This article originally appeared in The Sun and was reproduced with permission.