The commission's decision on how many votes to throw out will determine whether Karzai wins outright, or if he will have to fight a runoff against Abdullah Abdullah, his main challenger.
A deputy campaign manager for Abdullah called Barakzai's resignation a move by the Karzai campaign to call into question the validity of the fraud investigations.
"Barakzai's resignation has direct connection to Karzai. It was Karzai's idea," Saleh Mohammad Registani said.
"Karzai is trying to bring the work of the ECC into question."
He said Barakzai was widely considered to be biased toward Karzai.
An official with Karzai's campaign rejected that the president or his supporters had exerted any influence on Barakzai.
"He is totally independent," said Arasalah Jamal, the campaign's liaison to the commission.
Jamal said he had been in a meeting where Barakzai had said he had not been informed of decisions on the investigation process just a few days before he quit.
Barakzai was appointed to his post on the ECC by the Afghan supreme court, whose judges were named to their posts by the president.
He has held a number of government posts, but none overtly political.
"The people of Afghanistan have the right to know who will be the next president"
Rangeen Dadfar Spanta, Afghan foreign minister
He has served as the head of the Supreme Court's Islamic verdict department and its documentation department and during Afghanistan's 2005 elections, he headed the media complaints commission, overseeing coverage of the election.
Karzai said in an interview with ABC television that Barakzai's resignation cast doubt on the work of the commission and that officials should "do everything now to remove those suspicions".
The president has pressed to have his 54.6 per cent first-round victory confirmed quickly, but the ECC's delay has given hope to Abdullah's camp.
Western ambassadors are backing the ECC, irritating Karzai, who has criticised unnamed "outside circles" for interfering in the Afghan election.
Meanwhile, Afghan politicians have expressed frustration over the delays to the poll results.
"The people of Afghanistan have the right to know who will be the next president and whether we will we go to a second round or not," Rangeen Dadfar Spanta, the Afghan foreign minister, was reported by Reuters as saying.
"To leave the people in this vacuum, this is not in our interest."
The protracted process has kindled tension between Karzai and his Western backers, left Afghanistan in political limbo and helped delay a decision by Barack Obama, the US president, on sending the extra troops his battlefield commander has said he needs.