A delay in a Senate committee vote on Tuesday handed Bush a political defeat, at least in the short term, and opened the possibility that the nomination could fail.
A few Republicans on the Senate Foreign Relations Committee joined Democrats in asking to delay a vote.
Both Democrats and Republicans had earlier predicted the Republican-controlled committee would vote along party lines to recommend Bolton for the job.
"The dynamic has changed," said Republican Senator Lincoln Chafee of Rhode Island. "A lot of reservations surfaced today. It's a new day."
The Bush administration stood by Bolton and called allegations of abusive personal behaviour unfounded. There was no indication on Tuesday that Bolton might withdraw his name, but it was clear his nomination was in some trouble.
"My own hope is that the president decides to nominate someone else for this important position of UN ambassador," Democratic Senator Barbara Boxer of California said after Tuesday's tense committee meeting.
"Surely there must be many other men and women who could fulfil this post with honour."
The Senate committee set no new date for a vote, but a delay of at least two weeks seemed likely.
Democrats plan to use the time to investigate new allegations that Bolton abused his authority and mistreated subordinates, and to look into his unusual request for the names of other US officials whose communications were secretly picked up by a US spy agency.
"Surely there must be many other men and women who could fulfil this post with honour"
California Democratic Senator
The decision to postpone a vote closed a rancorous session in which some Democrats bluntly questioned Bolton's truthfulness and repeatedly appealed for more time to investigate him.
"We'll all have to trust each other," said Senator Richard Lugar of Indiana, the committee's Republican chairman, in sealing the unanimous agreement.
Republicans hold a 10-8 majority on the panel, and Lugar had sounded confident early in the session that he had the votes to prevail. He pushed hard for an immediate vote, over loud objection from Democrats.
"What's happening is that some Democrats on the committee are continuing to raise unfounded allegations"
White House press secretary
"Shocking," muttered Senator John Kerry of Massachusetts, as Lugar tried to hustle the process along.
The tide turned when Ohio Republican Senator George Voinovich spoke for the first time. He did not attend Bolton's two-day confirmation hearing last week, but had been presumed to be a supporter.
"I don't feel comfortable voting today," Voinovich said.
Republicans Chafee and Chuck Hagel of Nebraska also expressed reservations about a quick vote, and Hagel warned that he might not support Bolton's nomination if it should move to the full Senate.
"What's happening is that some Democrats on the committee are continuing to raise unfounded allegations," White House press secretary Scott McClellan said.
"We believe John Bolton has addressed all these issues. He's testified for more than eight hours. He's responded to many questions in writing as well, and we look forward to addressing any questions the committee members continue to have."
Bolton is currently the State Department's arms control chief. If approved, he would replace John Danforth as the US ambassador to the United Nations.