The original case was filed in December by Tamara Green, who has accused Cosby of assaulting her in the 1970s.
The two new plaintiffs are Therese Serignese and Linda Traitz.
The women claim the comedian publicly called them liars through statements issued by his representatives. Cosby is facing a series of accusations, which he has called “unsubstantiated”.
The women’s lawyer, Joseph Cammarata, said the civil action allows them to have their allegations heard now criminal statutes of limitation have expired.
The statute of limitations restricts the length of time in which legal action can be taken after an alleged crime has been committed.
The amended complaint was filed on Monday in the US district court in Springfield, in western Massachusetts, where Cosby has a home in Shelburne Falls.
“The lawsuit provides an opportunity for women who claim to have been harmed to have their day in court in a forum where the truth can be tried,” Mr Cammarata said.
Cosby’s publicist David Brokaw and lawyer Martin Singer did not immediately respond to requests for comment on Monday.
Mr Singer has said previously that he expected to prevail in the original defamation case.
Ms Green, who first spoke out regarding her allegations in 2005, said in her claim that Mr Brokaw had made statements intended to expose her to public contempt and ridicule.
Ms Serignese and Ms Traitz, from Florida, allege in the amended complaint that when they came forward in November last year, Mr Singer issued responses for Cosby that said they were lying.
Best known as Dr Cliff Huxtable on The Cosby Show from 1984 to 1992, the 77-year-old comedian is facing a number of allegations dating back to the 1970s.
The accusations, which he has described as “fantastical” and “uncorroborated”, have led to some of his stand-up shows being called off and the cancellation of some TV projects.