Separately, US District Judge Alvin Hellerstein, who is overseeing the case, denied a request by Flores to be released from jail due to health concerns ahead of a trial scheduled for next year.
Maduro, 63, and Flores, 69, were captured on January 3 in their Caracas home during a US military operation ordered by President Donald Trump, and were flown to New York to face drug charges against them that were announced that same day.
A superseding indictment filed in Manhattan federal court on Thursday added the torture count to the existing criminal case.
Maduro and Flores each would face a mandatory minimum 50 years in prison if convicted of all the charges.
They have pleaded not guilty to the original charges but have not yet entered pleas to the new indictment, which detailed alleged torture against nine people including three US citizens.
In a statement, Maduro's lawyer Barry Pollack said, "President Maduro denies the charges in the new indictment and looks forward to his day in court."
Flores' lawyers Mark Donnelly and Andres Sanchez in a statement said their client denies the allegations.
They questioned why prosecutors only revealed the indictment, which court records showed was returned under seal by a grand jury on Tuesday, shortly before Thursday's hearing.
"We look forward to fully responding to the allegations in court at the appropriate time," Donnelly and Sanchez said.
Since Maduro's capture, Venezuela has boosted co-operation with the US under the leadership of his former vice-president Delcy Rodriguez.
Relations between the United States and oil-rich Venezuela were tense during the socialist Maduro's 13 years in power.
US officials and human rights groups have long accused Venezuelan officials of torturing detainees but Thursday's indictment marked the first time Maduro faced criminal charges over the allegations.
The indictment alleged that thousands of Venezuelans were threatened, arrested, tortured or killed at the hands of the state between 1999 and 2025.
Before Maduro became president in 2013, both he and Flores held positions in the late Venezuelan president Hugo Chavez's government.
"Maduro, Flores and other high-ranking members of their inner circle used a violent system of torture and repression to maintain their power," Jamie McDonald, the top federal prosecutor in Manhattan, told reporters outside the courthouse after Flores' bail was denied.
Flores was Venezuela's attorney general and led its National Assembly before becoming first lady.
Many of the incidents of alleged torture described in the indictment occurred during times of political unrest in Venezuela or in response to perceived threats against Maduro.
In one instance, prosecutors said, Maduro in 2013 ordered the arrest of a Venezuelan military officer he suspected of planning a coup.
Members of Venezuela's military counterintelligence directorate then interrogated the officer while shocking the individual with a live electric wire about 20 times and hung the individual by the hands and feet for several minutes at a time, prosecutors said.
Flores' lawyers had argued their client should be released on bail because she needs a cardiac procedure and wants to recover in a more comfortable environment.
The lawyers proposed moving her to a residence in the New York City area with round-the-clock private security, and said she is not a danger to the community.
In denying the motion for bail, Hellerstein decided the evidence against Flores appeared strong.
The judge also said Flores, who wore a dark prison T-shirt and a headset to listen to a simultaneous Spanish translation of the proceedings, appeared to be relatively healthy.
"For whatever it's worth, her appearance today is no worse and perhaps a little better than when I first met with her on her arraignment," Hellerstein said, referring to her first appearance in court on January 5.
"She was still bandaged up from the military invasion," Donnelly said.
Prosecutors in the Manhattan US Attorney's Office opposed Flores' release.
They said Flores has received adequate medical care at the detention centre, and if released poses an "extreme" risk of flight and danger to the public, including possible witness intimidation.
