Rumours suggesting that the pair were dating have surfaced in recent months, with the House actress previously denying any romantic attachment to the singer.
The pair were most recently spotted together at a mutual friend's birthday party in New York, prompting further rumours.
However, Timberlake's spokesperson told People: "They are friends and are not romantically involved."
They added that the Social Network star is currently single, saying: "Nor is Mr Timberlake romantically involved with anyone."
Wilde had previously reassured her Twitter followers by saying: "Cool it, honeybadgers. We are just friends and have been for years."
Timberlake broke up with his long-time girlfriend Jessica Biel in March.