The couple were joined by Justin's mum and dad as they marked the Total Recall actress's 32nd birthday with dinner at The Forge restaurant and wine bar.
Justin, 33, even serenaded his wife of 26-months with a rendition of Happy Birthday.
"He sang a few lines of Happy Birthday, but nothing over-the-top," a source told the New York Post. "They looked extremely happy and comfortable."
On her special day Jessica tweeted her fans, thanking them for their kind wishes.
"Thanks for the birthday love!" she wrote. "You guys know how to make a girl feel good. Or woman, I guess... Or close to middle aged woman? Ah, forget it."
Justin and Jessica, who married on 19 October 2012 at the Borgo Egnazia resort in Fasano, Italy, became subject to speculation that their marriage was on the rocks in January.
Sources suggested that the pair were disagreeing over Justin's long tour absences and his apparent reluctance to start a family.
The rumours increased when Jessica attended the Oscars alone on Sunday 2 March. The Minnesota-born brunette dazzled in a Chanel Couture gown, but cut a lonely figure as she walked solo on the red carpet.
Justin was keen to support his wife on the night, however, retweeting a fan during the event who posted "@jtimberlake Your wife @JessicaBiel Looks gorgeous!"