He reiterated his call for a full investigation of the Nov 26 incident and said the relationship with Pakistan was critical to the security interests of both nations.
The Nato air strike sparked fury in Pakistan and complicated US-led efforts to ease tensions in relations with Islamabad, still seething at a secret US raid in May that killed al Qaeda leader Osama bin Laden, and to stabilize the region before foreign combat troops leave Afghanistan in 2014.
"The president made clear that this regrettable incident was not a deliberate attack on Pakistan and reiterated the United States' strong commitment to a full investigation," the White House said.
"The two presidents reaffirmed their commitment to the US-Pakistan bilateral relationship, which is critical to the security of both nations, and they agreed to stay in close touch," the statement added.
Obama's phone call came on the eve of an international conference in Germany on the future of Afghanistan.
Pakistan is boycotting the conference because of the Nato air strikes.
On Saturday, US secretary of state Hillary Clinton spoke by phone with Pakistani Prime Minister Yusuf Raza Gilani to offer condolences.