In a meeting with families of the 16 Afghan civilians killed on Sunday in southern Afghanistan, Mr Karzai said the delegation he sent to investigate the shootings did not receive the support they expected from American officials.
His criticism comes as the soldier suspected of carrying out the killing spree was said to be flying back to the US amid concerns for his safety.
Afghanistan was also hit by further tragedy yesterday as it emerged that 16 people – including two young girls – were killed in a helicopter crash. Twelve Turkish soldiers aboard the military helicopter died along with four Afghan civilians on the ground, including the two girls, according to the Afghan interior ministry.
A statement from the Turkish military said the cause of the accident was not known although there were no reports of insurgent activity in the area.
A house hit by the helicopter collapsed and rescue workers were searching through the rubble to determine whether there were any more casualties.
Turkish Foreign Minister Ahmet Davutoglu confirmed that a Turkish helicopter crashed near Kabul.
During the Afghan president’s meeting with the relatives of the victims of the shooting they insisted there must have been more than one soldier involved and claimed that they did not receive all the information they asked for from Americans.
Previously Afghan officials had said that there was surveillance video that was kept from them.
Mr Karzai said: “This has been going on for too long. This is by all means the end of the rope here. This form of activity, this behaviour, cannot be tolerated. It’s past, past, past the time.”
The Afghan leader stressed that he wanted a good relationship with the US but that it was becoming increasingly difficult. He insisted that the US needs to respect Afghan culture and laws.
The US staff sergeant suspected of the killings is accused of slipping out of his base before dawn on Sunday and sneaking into the homes of three Afghan families, shooting 16 of them dead and burning some of the bodies. Another five people were wounded.
The soldier has not been identified, but officials have said the 38-year-old is based in Washington state. He was transferred late on Wednesday to a facility in Kuwait and is believed to have now been flown to a military prison in the US.
The soldier’s lawyer, John Henry Browne, told reporters that the day before the rampage, he saw his friend’s leg blown off.
It is not clear whether the incident might have helped prompt the horrific middle-of-the-night attack.
The suspect had been injured twice during his three previous deployments to Iraq, and did not want to go to Afghanistan to begin with, Mr Browne added.
He declined to release his client’s name, citing concerns for the man’s family, but he said the soldier has two young children, ages three and four.
On Thursday the American campaign in Afghanistan suffered two punishing blows as the Taliban announced they were breaking off talks with the US and Mr Karzai tried to speed up the transfer of security responsibilities to Afghan forces by calling on international forces to pull out of rural areas.
Afghan officials said Mr Karzai wanted the pullback to start now, but US officials said he did not tell visiting Defence Secretary Leon Panetta that it should happen immediately. Mr Karzai said he had spoken to US President Barack Obama yesterday and added: “I insist on this issue. The fight is not in the villages, not in the houses of Afghanistan.”