Francis said that until he sees proof that Bishop Juan Barros was complicit in covering up the sex crimes of the Reverend Fernando Karadimas, such accusations against Mr Barros are "all calumny."
The pope's remarks drew shock from Chileans and immediate rebuke from victims and their advocates.
They noted the accusers were deemed credible enough by the Vatican that it sentenced Karadima to a lifetime of "penance and prayer" for his crimes in 2011.
A Chilean judge also found the victims to be credible, saying that while she had to drop criminal charges against Karadima because too much time had passed, proof of his crimes was not lacking.
"As if I could have taken a selfie or a photo while Karadima abused me and others and Juan Barros stood by watching it all," tweeted Mr Barros' most vocal accuser, Juan Carlos Cruz.
"These people are truly crazy, and the pontiff talks about atonement to the victims. Nothing has changed, and his plea for forgiveness is empty."
The Karadima scandal dominated Francis' visit to Chile and the overall issue of sex abuse and church cover-up was likely to factor into his three-day trip to Peru that began late on Thursday.
Karadima's victims reported to church authorities as early as 2002 that he would kiss and fondle them in the Santiago parish he ran, but officials refused to believe them.
Only when the victims went public with their accusations in 2010 did the Vatican launch an investigation that led to Karadima being removed from ministry.
Francis had sought to heal the wounds by meeting this week with abuse victims and begging forgiveness for the crimes of church pastors. But on Thursday, he struck a defiant tone when asked by a Chilean journalist about Mr Barros.
"The day they bring me proof against Bishop Barros, I'll speak," Francis said. "There is not one shred of proof against him. It's all calumny. Is that clear?"
Anne Barrett Doyle, of the online database BishopAccountability.org, said it was "sad and wrong" for the pope to discredit the victims since "the burden of proof here rests with the church, not the victims - and especially not with victims whose veracity has already been affirmed."
"He has just turned back the clock to the darkest days of this crisis," she said in a statement. "Who knows how many victims now will decide to stay hidden, for fear they will not be believed?"
Indeed, Catholic officials for years accused victims of slandering and attacking the church with their claims. But up until Francis' words on Thursday, many in the church and Vatican had come to reluctantly acknowledge that victims usually told the truth and that the church for decades had wrongly sought to protect its own.
German Silva, a political scientist at Santiago's Universidad Mayor, said the pope's comments were a "tremendous error" that will reverberate in Chile and beyond.
Patricio Navia, political science professor at Diego Portales University in Santiago, said Francis had gone much further than Chilean bishops in acknowledging the sexual abuse scandal, which many Chileans appreciated.
"Then right before leaving, Francis turns around and says: 'By the way, I don't think Barros is guilty. Show me some proof,'" Mr Navia said, adding that the comment will probably erase any good will the pope had won over the issue.
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