The "Suit & Tie" singer, who wore a large black fedora and dark scarf (no tie!) for the snazzy, casino-themed affair, held at his Hell's Kitchen restaurant Southern Hospitality, arrived at the event around 8:30 p.m. with Biel, 31, by his side.
The Playing for Keeps actress made a fashion statement of her own, donning a cream-colored silk, collared shirt with a bow tie, black crocheted pants and a black Chanel bag.
The pair mixed and mingled with the star-studded crowd throughout the night, though Timberlake, 32, would often "come over and kiss her and talk to her one-on-one," one eyewitness tells Us.
The crooner spent much of the evening chatting with his "Suit & Tie" collaborator, Jay-Z, the onlooker said. At one point, the musical pair even escaped to the downstairs casino to play blackjack while Biel and a female friend tried their hand at roulette (proceeds from the night went to Timbaland's charity, the Always Believing Foundation).
When Timbaland -- boasting a dapper bow tie -- and wife Monique arrived around 10 p.m., Biel congratulated the host and hostess for the Neuro Drinks-sponsored fete. DJ Ani Quinn provided the music for the low-key bash.
"What a great party! Thanks for having us," the actress said, according to the onlooker.
Beyonce popped by about an hour later, dressed down in wedge sneakers, a bomber jacket and no makeup, coming directly from a rehearsal at the Brooklyn Navy Yard.