U.S. officials said the strike may have been based on outdated intelligence, and an investigation into the incident is underway.
The pope said he was particularly close to the families of victims killed in attacks that have struck schools, hospitals and residential areas during the conflict.
He also expressed concern about the impact of the fighting in Lebanon, where aid groups have warned the escalating conflict could trigger a humanitarian crisis.
Christian communities in southern Lebanon are of particular concern to the Vatican, as they have long been seen as an important presence for Christians across a largely Muslim region.
For much of the two weeks since the conflict began, Leo has limited his public comments to broader appeals for peace and dialogue while avoiding direct references to the U.S. or Israel – a stance consistent with the Vatican’s longstanding tradition of diplomatic neutrality.
Some Catholic leaders, however, have taken a more direct stance on the conflict.
Cardinal Robert McElroy, the archbishop of Washington, described the war as morally unjustifiable, while Chicago Cardinal Blase Cupich criticized the White House for sharing social media posts about the war that included video game-style imagery.
Meanwhile, Vatican Secretary of State Cardinal Pietro Parolin rejected Washington’s characterization of the fighting as a "preventive war," but said the Holy See continues to keep lines of communication open with all sides.
"The Holy See speaks with everyone," Parolin said. "When necessary we speak also with the Americans, with the Israelis and show them what to us are the solutions."
The Associated Press contributed to this report.