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."
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.