In an interview with the BBC, former foreign minister Abdullah Abdullah said the Independent Election Commission was corrupt and helping to rig the vote in favour of Karzai.
Voters would feel cheated with a fraudulent vote and that would lead to more instability in the conflict-torn country, he said.
"It's not independent at all; it's on President Karzai's side," he said. "It has been corrupt, and their malpractice is now widespread.
"It's not for the good of the country that somebody who commits massive fraud rules the country for five years."
Karzai has defended the Aug. 20 election as honest but the standoff has alarmed the United States and its allies, whose troops are involved in an increasingly unpopular mission.
His campaign spokesman Wahid Omar told BBC radio the allegations had made the work of the investigators more confusing, and warned against local or international attempts to influence voting authorities.
"We actually believe that most of the remarks that have been made should not be taken as the reality," he said.
"These are all rumours and I think we should not base our statements on rumours that we have no way to verify."
The election is a key element in Western plans to bring stability to Afghanistan and to prevent militants from using the country as a base for attacks across the globe.
More than 100,000 foreign troops in a U.S.-led coalition are deployed in the country fighting the Taliban, who were ousted from power in 2001 but whose insurgency is growing stronger.
U.S. and U.N. officials say they are waiting for the final tally and the outcome of fraud investigations before concluding whether the result is fair.
Abdullah was quoted as saying he still wanted a fair result to be found through peaceful channels.
"I'm not talking about just my own supporters, but those who cast their vote for Mr Karzai," he told BBC radio. "Their vote is now part of the fraud. And on top of that, a fraudulent outcome: illegitimate rule for another five years.
"I think this in itself is a recipe for instability in this country."
Preliminary election results issued on Tuesday gave Karzai more than 54 percent of valid votes tallied, putting him above the 50 percent threshold needed to avoid a runoff with Abdullah.
