The allegation that soldiers violated the neutrality of a medical facility follows the in the country's north last week.
Nearly eight years after the U.S.-led coalition invaded to oust the Taliban, foreign forces are working to persuade the population to support the Afghan government. But civilian deaths and intrusive searches of homes have bred resentment.
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The Swedish Committee for Afghanistan said the U.S. Army's 10th Mountain Division entered the charity's hospital without permission to look for insurgents in Wardak province, southwest of Kabul, according to the charity's country director, Anders Fange.
"This is simply not acceptable," he said.
The U.S. troops came to the hospital looking for Taliban insurgents late at night last Wednesday, Fange said. He said they kicked in doors, tied up four hospital employees and two family members of patients, and forced patients out of beds during their search.
When they left two hours later, the unit ordered hospital staff to inform coalition forces if any wounded militants were admitted, and the military would decide if they could be treated, Fange said.
The staff refused, he said. "That would put our staff at risk and make the hospital a target."
The charity said on its Web site that the troops actions were not only a violation of humanitarian principles but also went against an agreement between NATO forces and charities working in the area.
"We demand guarantees ... that such violations will not be repeated and that this is made clear to commanders in the field," a statement said.
Navy public affairs officer Lt. Cmdr. Christine Sidenstricker confirmed that the hospital was searched last week but had no other details. She said the military was looking into the incident.
"We are investigating and we take allegations like this seriously," she said. "Complaints like this are rare."
Violence has surged across much of Afghanistan since President Barack Obama ordered 21,000 more U.S. troops to the country this year. Two foreign troops were killed Sunday when their patrol hit a roadside bomb in the country's south, NATO said without giving their nationalities. Three civilians also died in a militant rocket attack on the capital.
NATO was also investigating reported civilian deaths in a U.S. air strike last week. Afghan officials said up to 70 people were killed in the early morning air strike Friday in the northern province of Kunduz after the Taliban hijacked two fuel tanker. After the trucks became stuck in the mud on the banks of a river, villagers came to siphon off gas and some were reported killed when an American jet dropped two bombs on the stolen tankers.
The increasingly violent Taliban have killed more Afghan civilians in bombings and other attacks. On Monday, the government said three insurgent rockets landed in the capital, Kabul, killing three people when one of them hit a house.
A United Nations report in July said the number of civilians killed in conflict in Afghanistan has jumped 24 per cent this year, with bombings by insurgent and air strikes by international forces the biggest single killers. The report said that 1,013 civilians were killed in the first half of 2009, 59 per cent in insurgent attacks and 30.5 per cent by foreign and Afghan government forces. The rest were undetermined.