Weinstein, 72, who is recovering from emergency heart surgery, on Wednesday entered his plea to committing a criminal sexual act in the first degree at a hearing before Justice Curtis Farber in a New York state court in Manhattan.
"Not guilty," Weinstein responded emphatically when asked for his plea to the felony charge.
Weinstein still faces two other criminal counts from an earlier indictment where he also pleaded not guilty, including another first-degree criminal sexual act charge and a third-degree rape charge.
He appeared in court in a wheelchair, wearing a dark suit and a blue tie, and with a large bandage on his right hand.
The new charge announced by Manhattan District Attorney Alvin Bragg accuses Weinstein of sexually assaulting a woman in a downtown Manhattan hotel between April 29 and May 6, 2006.
"Thanks to this survivor who bravely came forward, Harvey Weinstein now stands indicted for an additional alleged violent sexual assault," Bragg said in a statement.
Weinstein's latest accuser has yet to be publicly identified.
"She will be fully prepared to speak her truth at trial to hold Mr. Weinstein accountable before a jury of his peers," her lawyer Lindsay Goldbrum said in a statement.
It was unclear whether a retrial of Weinstein would include the three charges he now faces.
Jurors had found Weinstein guilty of rape and sexual assault in February 2020, less than two-and-a-half years after sexual misconduct accusations made his case the impetus for the #MeToo movement.
But the state's highest court, the Court of Appeals, threw out Weinstein's conviction and 23-year sentence in April, saying he did not get a fair trial because the judge allowed testimony from women who Weinstein was not charged with assaulting.
Weinstein has denied having non-consensual sexual encounters with anyone. His original trial judge, James Burke, was not reappointed to the bench after his term expired at the end of 2022.
The new charge was announced two months after prosecutors said they were investigating additional alleged "violent sexual assaults" by Weinstein, after new accusers came forward.
Farber at the time set a tentative November 12 retrial date, but prosecutors said on Wednesday they would likely not be ready until January.
Weinstein's lawyer Arthur Aidala said his client wants to go trial "as soon as humanly possible".
He later told reporters that Weinstein was "vehemently opposed" to adding the new charge to the original case, and wants a retrial on the original charges as soon as possible.
Farber did not set a new trial date, but said he would proceed when both sides are ready.
Weinstein remains in custody, because he was convicted separately in 2023 in California for the 2013 rape of an actress in Los Angeles and sentenced to 16 years in prison.
Weinstein's lawyers have said he is beset with health problems. He was rushed to the hospital from New York City's Rikers Island jail on September 8 to undergo heart surgery.
Once among Hollywood's most powerful people, Weinstein co-founded the Miramax studio, whose hits included Shakespeare In Love and Pulp Fiction.
Weinstein's film studio filed for bankruptcy in March 2018, imploding after the original accusations of sexual misconduct.
In the New York trial, prosecutors portrayed Weinstein as a serial predator who manipulated women with promises of career advancement in Hollywood, coaxed them into hotel rooms or private apartments, and then overpowered and attacked them.
