"How many of you didn't hang out with questionable folks in high school?" she asked fellow Republicans at a GOP picnic in southern Delaware on Sunday.
"There's been no witchcraft since. If there was, Karl Rove would be a supporter now," O'Donnell jokingly assured the crowd.
Rove, the former GOP strategist and adviser to President George W. Bush, has suggested that O'Donnell's win in last week's GOP primary cost Republicans a chance to retake the Senate seat long held by Democrat Joe Biden before he was elected vice president.
O'Donnell, a conservative Christian activist, rode a surging tide of tea party activism to an upset victory over GOP moderate Michael Castle, Delaware's longtime congressman and former two-term governor. She faces Democratic county executive Chris Coons in November.
O'Donnell's comments about witchcraft were made during a 1999 taping of comedian Bill Maher's "Politically Incorrect" show.
"I dabbled into witchcraft. I never joined a coven," she said on the show, a clip of which hit the Internet as O'Donnell canceled Sunday appearances on two national news shows, citing commitments to attend church and the GOP picnic in Delaware.
"I hung around people who were doing these things. I'm not making this stuff up. I know what they told me they do," O'Donnell told Maher.
"One of my first dates with a witch was on a satanic altar, and I didn't know it. I mean, there's little blood there and stuff like that," she said. "We went to a movie and then had a little midnight picnic on a satanic altar."
Russ Murphy, executive director of the 9-12 Delaware Patriots, a group that joined in the tea party effort to propel O'Donnell to Tuesday's primary victory, said the focus on her comments about witchcraft was just another attempt by pundits and political opponents to discredit her.
"They're going to be pulling for straws from the sky to do anything to stop this momentum, and they don't realize it's not going to work," he said.
O'Donnell's victory in the primary came after a bruising campaign in which her supporters and Castle's, led by state GOP chairman Tom Ross, traded attack ads, with Ross saying O'Donnell was a liar and a fraud who couldn't be elected dogcatcher.
Ross did not attend Sunday's Sussex County Republican Committee picnic. Sussex County GOP chairman Ron Sams said Ross was in Washington trying to drum up support from the national GOP campaign committees.
Despite her improbable primary victory, O'Donnell sounded upbeat about her chances in November.
"We're going to win this by uniting the party," she told supporters. "I'm very confident that we're going to win this election."