The ad, launched by Keep America Safe, an advocacy group led by Cheney, criticizes Holder for not disclosing details about Justice Department lawyers who have previously defended alleged terrorists.
The video, which surfaced last week, brands the lawyers as the "Al Qaeda 7" and ridicules the Justice Department as the "Department of Jihad." The narrator questions, "Who are these government officials?...Whose values do they share?"
Justice Department spokesman Matthew Miller has called it "offensive" that the patriotism of agency lawyers is questioned by Cheney's group.
But a growing chorus of former Bush administration officials, including former Attorney General Michael Mukasey, also say the video goes too far. Mukasey called the ad's accusations "shoddy and dangerous."
Keep America Safe is not backing down, insisting the questions it raises are responsible and valid.
"The American people have a right to know who in the Department of Justice is setting policy regarding detention of terrorists and related national security issues," Aaron Harison, executive director of Keep America Safe, told ABC News.
"Lawyers in private practice have the right to volunteer 'pro bono' to defend terrorists. However, membership in the legal profession does not immunize a person from questions or criticism of their prior actions," he said.
Cheney, the normally outspoken daughter of former vice president Dick Cheney, did not respond to ABC News' requests for an interview.
Republican lawmakers have pressed Holder for greater detail on the lawyers' backgrounds and the policy decisions and specific detainee cases with which they may be involved. However, many openly disagree with the ad's provocative nature.
"It's wrong to criticize attorneys who represent alleged terrorists," Mukasey wrote in a Wall Street Journal op-ed Wednesday.
Mukasey said it is "prudent" to question whether such lawyers may be influenced by an outside agenda and such questions are within the purview of Congress' oversight responsibility.
However, he said the lawyers should not "automatically to be identified with their former clients and regarded as a fifth column within the Justice Department."
High-Profile Republicans Defend Lawyers Representing Detainees
Several other high-profile conservatives have joined Mukasey in criticizing Cheney's group's characterization of the former Guantanamo Bay detainee advocates.
Former federal prosecutor, Georgia Republican congressman and Libertarian presidential candidate Bob Barr, attacked Liz Cheney herself for what he called "disgraceful pandering to the extreme right."
"In Cheney World, lawyers who dared to provide legal counsel for those facing such serious charges as committing acts of terrorism should be forever thereafter barred from serving in any government job; and also probably should be branded on their forehead with a scarlet "T" (for "terrorism lover")," Barr wrote in a column in the Atlanta Journal Constitution.
Keep America Safe insists it is not calling for the lawyers' resignations or removal from office, only greater transparency. But the video's implications have irked many conservatives.
On Sunday, the Brookings Institution released a statement signed by 22 former Republican administration officials and lawyers calling the attacks "shameful" and "destructive."
"To suggest that the Justice Department should not employ talented lawyers who have advocated on behalf of detainees maligns the patriotism of people who have taken honorable positions on contested questions and demands a uniformity of background and view in government service from which no administration would benefit," it reads.
Among the signatories is former independent prosecutor Kenneth Starr, who led the investigation into President Bill Clinton's sex scandal, and former acting attorney general Peter Keisler in the administration of President George W. Bush.
During that Bush administration, the Justice Department hired at least two lawyers who defended terrorist suspects in private practice. Trisha Anderson represented 13 Yemeni detainees while with the firm Covington & Burling and Varda Hussain represented three Egyptian detainees while with the firm Venable.
It does not appear that Liz Cheney or other Republicans objected to those appointments.
Holder Critics Call for Greater Transparency
"Somebody who was advocating for the release of terrorists... that kind of person is unlikely to be able to put in place the policies that are necessary to keep us safe," Cheney said on Fox News on March 4.
The Justice Department refutes that charge and says it does not permit its lawyers' previous affiliations to interfere with their government duties. In several instances, attorneys have been recused from cases involving specific detainees with whom they may have worked, according to a recent letter from Holder to Republican senators.
Republicans and Cheney's group are pressing Holder for information on the specific cases that have involved recusals and on which policy decisions the lawyers have worked.
Holder is expected to testify before a Senate oversight panel on March 23.
ABC News' Jake Tapper contributed to this report.