Karzai's comments on the Wednesday airstrike in Logar province, about 50 miles from Kabul, came in a statement in which he said a thorough investigation of the airstrike would be conducted. He returned early from a conference in China to deal with the attack, The Washington Post reported.
"NATO operations that inflict human and material losses to civilians can in no way be justifiable, acceptable and tolerable," Karzai said in the statement.
NATO "is taking the allegations seriously," said Martyn Crighton, a spokesman for NATO forces.
"As a part of that process, the commander has dispatched a team to gather facts, meet with Afghan officials and try to determine what happened," he said.
Within hours of the airstrike, two suicide bombers attacked a Kandahar market, killing at least two civilians.
The Post said the two incidents made Wednesday one of the bloodiest days of the war for Afghans.
Attacks by insurgents and coalition forces in Afghanistan Wednesday killed at least 40 people, including civilians, officials said.
"While the figures are preliminary, it is fairly clear this is the deadliest day of the year for civilians," said James Rodehaver, acting head of the U.N. human rights division in Afghanistan.
Another suicide attack Wednesday in northern Afghanistan killed one Afghan, officials said.
Also Wednesday, ISAF officials have begun an investigation to determine whether insurgent fire brought down a helicopter in eastern Afghanistan, killing two soldiers, The Wall Street Journal said.
Karzai's criticism came the same day U.S. Defense Secretary Leon Panetta said during a visit to Kabul U.S. officials are losing patience with Pakistan providing havens for terrorists.
"We are reaching the limits of our patience, and for that reason it is extremely important that Pakistan take action to prevent this kind of safe haven," Panetta said at a news conference with Afghan Defense Minister Abdul Rahim Wardak.
Panetta called on Pakistan to do more to root out the al-Qaida-linked Haqqani network from havens in a federally administered tribal area of Pakistan.
"We will continue to make it clear that it is an intolerable situation," he said. "We will take whatever steps necessary to protect our forces -- that's a principle that we always stand by. To make that happen we have to have the cooperation of Pakistan to take steps to control the Haqqani threat on their side of the border."