"General McChrystal is doing everything humanly possible to avoid civilian casualties," Gates said.
McChrystal, himself, addressed Afghans directly on Monday in a video posted online and translated into Dari and Pashto.
"I have made it clear to our forces that we are here to protect the Afghan people. I pledge to strengthen our efforts to regain your trust to build a brighter future for all Afghans," he said in the video message.
"Most importantly, I express my deepest, heartfelt condolences to the victims and their families. We all share in their grief and will keep them in our thoughts and prayers."
Gates, meanwhile, said that efforts to fight a resurgent Taliban were being complicated by the group's use of "civilians for cover".
|Taliban fighter tells Al Jazeera why he fights Nato forces and that Afghans back the Taliban
That point was echoed by Anders Fogh Rasmussen, the Nato secretary-general, who said the Taliban uses "women and children, innocent civilians, as a human shield".
Rasmussen expressed his condolences over the civilian deaths and said Nato troops were doing everything in their power to limit civilian casualties, and that despite the latest tragedy, those efforts were working.
"There will be bad days, included when we cause unintended civilian casualties," Rasmussen said, but added: "During the recent year, our troops in Afghanistan have succeeded in reducing the number of civilian casualties significantly."
In recent months, Nato has limited air strikes and tightened rules of engagement on the battlefield to try to protect Afghan civilians and win their loyalty from the Taliban.
Sunday's air raid on a major road near Uruzgan's border with Day Kundi province was the worst case of civilian deaths in Afghanistan in months and the Afghan cabinet condemned it as unjustifiable.
The ministers called for Nato to "exercise maximum care before conducting any military operation" to avoid further civilian casualties.
The cabinet said 27 civilians were killed, including four women and a child, and 12 people were injured.
Civilian casualties have caused friction between the government and foreign forces, with Hamid Karzai, the Afghan president, repeatedly calling for Nato to do more to protect civilians during military operations.
"We need to reach the point where there are no civilian casualties. Our effort and our criticism will continue until we reach that goal," Karzai said during the Afghan parliament's opening session in Kabul on Saturday.
Sunday's incident was the second time in nine days that Nato has apologised for killing civilians. On February 14, two US rockets hit a home outside Marjah, killing 12 people, including six children.
The latest civilian deaths come as Nato and Afghan troops continue their offensive against the Taliban in southern Helmand province's Marjah town.
Although the air raid was not related to the Marjah offensive, civilian casualties undermine Nato's goal of turning back the Taliban and winning the confidence of the Afghan people as it employs a new strategy that stresses protecting civilians over routing fighters as quickly as possible.
Admiral Mike Mullen, the chairman of the US joint chiefs of staff, has also offered his "heartfelt condolences" to the families of those killed on Sunday for the "terrible loss of innocent civilians".
He noted that McChrystal had apologised to Karzai immediately following the incident.