A judge rejected Camille Cosby's attempts to avoid testifying, after she claimed she was protected by a marital disqualification law.
The women claim Mr Cosby portrayed them as liars after they went public with their allegations against him.
He filed a countersuit alleging their claims were only for financial gain.
The 78-year-old is also seeking unspecified monetary damages and said the women inflicted emotional distress.
Mrs Cosby is also her husband's business partner and the couple have been married for more than 50 years.
Joseph Cammarata, a lawyer for the women, argued last month he should be able to question Mrs Cosby because he believed she had "information that's relevant to the litigation in this matter".
As well as throwing out her marital status disqualification attempts, Judge Magistrate David Hennessey also rejected claims the value of her evidence would be undermined by the  "undue burden" it would cause her.
"The arguments do not outweigh the potential significance of Mrs Cosby's testimony, nor has Mrs Cosby provided any authority that they do," he said.
The comedian had also tried to prevent his wife having to testify by having his lawyers argue it was an attempt to put pressure on him by causing them both "shame and embarrassment".
Mr Cosby's accusers' legal claim alleges he and his representatives sought to tarnish their reputations after they went public with their claims of sexual misconduct.
Mr Cosby has repeatedly denied the accusations.
His counter legal claim contends the women "engaged in a campaign to assassinate" his reputation and character.
He has, however, admitted in court proceedings he obtained and gave women sedatives over the years, but he maintained the women took the drugs willingly.
Last week, prosecutors in Pennsylvania charged the comedian with an alleged sexual assault in 2004. It is the first time Mr Cosby has been charged with any offence after months of accusations by dozens of women.
He is currently on $1m (Â£675,000) bail.
Bill Cosby's wife has been ordered to give evidence in a defamation case filed by seven women accusing the comedian of sexual assault.