Shimaa Abdelfadeel and Hebba Aref were told by campaign volunteers that they could not sit in the seats they had chosen at the event in Detroit, Michigan, on Monday, as they were wearing hijabs.
The staffers' actions sparked accusations of Islamophobia in the Obama team, which has repeatedly had to deal with false claims the Ilinois senator and Democratic presidential candidate is a Muslim.
Mr Obama said the volunteers' actions were "unacceptable and in no way reflect any policy of my campaign".
"I take deepest offence to and will continue to fight against discrimination against people of any religious group or background," he said.
"I reached out to Ms Aref and Ms Abdelfadeel this afternoon," Mr Obama said in a statement. "I spoke with Ms Abdelfadeel, and expressed my deepest apologies for the incident that occurred."
He said Ms Abdelfadeel had accepted his personal apology, which came after an earlier apology from his campaign team over the blunder.
He is reported to have left a phone message for Ms Aref, who had earlier said she was deeply hurt by the volunteers' actions.
"I was coming to support him, and I felt like I was discriminated against by the very person who was supposed to be bringing this change," she told website Politico.com.
"The message that I thought was delivered to us was that they do not want him associated with Muslims or Muslim supporters," she said.
The Council on American Islamic Relations (CAIR) had called for a personal apology from Mr Obama to help redress the grievance and also combat growing anti-Islamic sentiment in the United States.
"We are extremely concerned about the level of Islamophobia in our society that would prompt other minorities to view Muslim supporters as potential liabilities," CAIR's national legislative director Corey Saylor said in a statement.
CAIR, the leading US group for Muslims' civil rights, has also urged Mr Obama to invite the women to the stage during a future campaign event.