"We are Catholics. Together, we are the church. … We will defend our right to practice our faith free from government coercion," the Catholic Association ad's narrator says over a photo of President Obama.
The group also announced a new poll finding that a majority of voters "oppose the notion that free birth control should be a federal priority."
Catholics for Choice, meanwhile, said Tuesday that more than 3,600 people have submitted comments to the Department of Health and Human Services in support of the mandate.
The group has also dismissed the bishops' two-week activism push, saying that warm-up events were poorly attended "even by the low standards that the bishops set themselves."
Announced in January, the policy means that most employers must cover contraception in their employees’ healthcare plans without charging a copay.
Churches are exempt, and employees of religiously affiliated institutions such as Catholic hospitals must be able receive contraception directly from their insurance companies, still without a copay.
The Obama administration made that concession in order to quiet some of the backlash that followed the announcement.
Supporters of the mandate made the case that women who choose to work in a Catholic hospital or school, which often employ and serve non-Catholics, should have access to the same birth control benefit as employees in an exclusively lay environment.
But in lawsuits filed against the administration, religious organizations demand exemptions for all their affiliated institutions.
There are now 23 cases pending against the mandate involving 56 plaintiffs, according to the Becket Fund for Religious Liberty.
The controversy took top billing at a recent assembly of U.S. Catholic bishops in Atlanta, where church leaders discussed the so-called "Fortnight for Freedom" meant to promote activism against the mandate.
A coalition of progressive Catholic groups responded with a letter arguing that "the use of contraception is a moral decision" to be left up to individuals.
"Our views on many important issues often diverge with the views of the Catholic hierarchy in the United States," it read. "Unfortunately, the bishops attempt to portray their views as representative of ours in public discourse."
Jon O'Brien, president of advocacy group Catholics for Choice, decried the tone of the meeting in Atlanta.
"The bishops’ discussion of religious freedom in Atlanta was a travesty, consisting of patting themselves on the back about their campaign to have the right impose their beliefs on the entire American population," he said in a statement. "The bishops just don’t get it."
The Catholic Association's 60-second ad will air nationwide on Fox News on Thursday only. The campaign cost "just under $100,000," according to a spokesman for the group, who added: "We will look at a broader buy later."