The singer and actress lodged a defamation action in the courts in Ireland over an article, photographs and quotes attributed to Total Recall actress Biel which were published last month.
The lawsuit was lodged in the High Court in Dublin against the magazine’s publishers, the German Bauer Media Group.
Timberlake and Biel have been married for two years.
It is understood the action is being taken in Dublin as the weekly celebrity magazine is sold in Ireland as well as across Europe.
The couple’s lawyer Paul Tweed, of Johnsons Solicitors, confirmed the papers have been lodged, but it could take more than a year for the case to go to trial.
“We have issued defamation proceedings on behalf of our clients, Justin Timberlake and Jessica Biel, in relation to an article and a headline in Heat magazine,” Mr Tweed said.
“We are suing in Ireland in relation to publication in all jurisdictions, as our clients are entitled to do.”
Timberlake and Biel’s defamation claims were lodged separately with the High Court on Wednesday.