"I once again highlight the need for all sides to meet their obligations to protect civilians," said Staffan de Mistura, the UN secretary-general's special representative.
The latest reports coincided with the publication on Monday by the whistleblower website Wikileaks of tens of thousands of classified US documents which cast a new light on civilians caught up in what it called "the true nature of this war".
A spokesman of the Afghan government said information that 52 civilians had been killed came from the country's intelligence service in the district.
Karzai strongly condemned the attack and asked Nato troops to prioritise the protection of civilians in their military campaign, his office said in a statement citing the same casualty figures for the attack.
Isaf, however, insisted that a joint investigation with the Afghan government had so far found no evidence of civilian deaths, while a provincial official suggested local residents could even have made it up.
"The villagers took the joint team to a graveyard in Regey village and they claimed that 35 people were buried there, but the graves seemed to be old," said Dawood Ahmadi, a spokesman for the provincial governor, referring to the village where the incident is supposed to have taken place.
"The team have not found any evidence to show that the civilians were killed," he said.
"They may have been lying but we are there to find out."
There are various compensation packages for civilians caught up in the fighting, but Isaf has reported many cases of wrongful claims.
An Isaf spokeswoman said the team was still in the area, trying to establish the truth.
"We take any civilian casualty very seriously but there was no report of operational activity in Regey," she said.
Isaf has a poor reputation among Afghans for investigating similar incidents.
In the worst attack of its kind, 140 civilians were killed in May last year in an Isaf air strike on a village in Western Farah province, among them 93 children and 25 women.
For days Isaf denied knowledge of the incident, and then suggested those killed were mostly insurgents, before admitting to a much lower casualty figure.
Wikileaks described a similar pattern with thousands of unreported civilian deaths in the near nine-year-old war.
A report by the Afghanistan Rights Monitor said nearly 1,100 civilians were killed in the first half of this year in Isaf-operations or Taliban attacks.
The UN said some 2,400 civilians were killed in conflict-related incidents last year.