The policy, announced by Attorney General Jeff Sessions in April, allows the prosecution of adults separately from children when families seek asylum at the border.
"I don't want children taken away from parents".
Four former first ladies also weighed in, led by Laura Bush, who wrote in a Washington Post op-ed: "This zero-tolerance policy is cruel. It is immoral. And it breaks my heart", she wrote in the publication.
Meanwhile, officials announced plans to erect tent cities to hold hundreds more children in the Texas desert where temperatures regularly reach 40C (105F).
The former first lady compared the facilities to the Japanese American internment camps of World War II, which she deemed "one of the most shameful episodes in US history".More news: Here's when Eid Al Fitr will be announced in UAE
Both Obama and her husband, former President Barack Obama, have largely remained on the sidelines since leaving the White House last January, speaking out against the Trump administration only on rare occasions and often in a veiled manner. When the families are separated, they are temporarily placed in detention centers.
The current bill proposed by House leadership would give amnesty to over 1.8 million children of illegal immigrants in exchange for modest border security funding.
As first lady, Bush created a formal platform of childhood education and literacy programs.
In the seven-minute tape, children who were reportedly aged four to 10, could be heard crying out for the parents.
The Trump administration argued it's trying to deter future illegal border crossings. Trump has made helping children the basis for her platform as first lady, called "Be Best".
The US government has said that during one recent six-week period, almost 2,000 minors were separated from their parents or adult guardians - a figure that stoked the firestorm. However, there is no law mandating the separation of families.
But she rejected criticism that the White House was attempting to use the policy as "leverage" to pass an immigration bill in Congress, and disagreed that Trump should reverse the policy.
"Nobody likes" breaking up families and "seeing babies ripped from their mothers' arms", Kellyanne Conway, a counsellor to the president, said.
"I live in a border state", Bush said.