Accurso's lawyer had argued that testifying at the Charbonneau commission would risk incriminating Accurso in connection with criminal charges he is facing.
Canada's top court has refused to hear his appeal, which means Accurso will be forced to testify.
The controversial construction magnate was arrested in April 2012 in a sweep by Quebec's anti-corruption unit (UPAC) and faces a string of charges, including fraud, conspiracy, influence-peddling, breach of trust and two counts of defrauding the government.
He was arrested once again a year ago, this time for his alleged role in a $3-million tax-evasion scheme.
Those charges have not been proven in court.
Accurso received a notice on July 18, 2013 to appear as a witness before the Charbonneau commission.
A date has not yet been set for his appearance at the inquiry.