According to an indictment unsealed on Tuesday, Noor Salman "did knowingly aid and abet" her husband's efforts to support the Islamic State group.
She has also been accused of misleading police investigators and FBI agents on the day of the 12 June 2016 attack.
Her family say she is a victim herself and innocent of the charges.
Ms Salman is charged with providing material support to a foreign terrorist organisation and obstruction of justice.
"She knew he was going to conduct the attack," federal prosecutor Roger Handberg told the judge.
Al Salman, an uncle of the 30-year-old woman, said that his niece was "simple and innocent" and was physically and mentally abused by her husband, Omar Mateen.
He asserted that she knew nothing about her husband's plan to attack Orlando's Pulse nightclub.
Mateen was killed in a shootout with police after killing 49 people and injuring 53 others in the worst mass shooting in modern American history.
During the attack, Mateen called police to pledge support for the Islamic State group.
A lawyer for Ms Salman said in court that she had no advance knowledge of the tragedy and she should be released from jail pending trial.
She was arrested on Sunday in the San Francisco area after moving there from Florida following the attack.
As she was led back to jail, she looked sadly back at her uncle, according to reporters inside the courtroom.
Another hearing is schedule for Wednesday to discuss possibly transporting her to Florida where she may face the federal charges there.
The widow of the man who killed 49 people at a Florida gay nightclub knew of the attack before it happened, a court in California has heard.