Asked about historic statements by Senator Roberts praising American conspiracy theorist Alex Jones, who spread the claim that the Sandy Hook massacre was staged by the US government as a pretence for stricter gun control, Mr Joyce said the issue was black and white.
"Sandy Hook was an outrageous murder of innocent people. It was no false flag event. Neither was 9/11," he told ABC Radio National on Thursday, referencing another conspiracy theory pushed by Jones.
As One Nation's rise in the polls sees them courting a more mainstream demographic, the party's associations with figures such as Jones and UK anti-immigration activist Tommy Robinson have come under greater scrutiny.
Mr Joyce was also asked about a social media post shared by Senator Roberts in 2024 of a mural that repeated an anti-Semitic trope about Jews controlling global finance and politics.
"For too long people have been blind to the Uniparty with Liberal and Labor pushing the same globalist UN-WEF policies," Senator Roberts wrote in the caption.
However Mr Joyce declined to be drawn in.
"I'm not here to start carpeting Malcolm. I'm not going to do that. If you wish to have a discussion with him, I'm sure you can have him on your show," he told ABC radio host Sally Sara.
Independent MP Allegra Spender said One Nation leader Pauline Hanson should demand Senator Roberts' resignation.
"It's shameful that one of One Nation's most senior MPs should endorse and share this notorious anti-Semitic slur," she wrote on X, formerly Twitter.
"Will Pauline Hanson or Barnaby Joyce ever hold him to account?"
Mr Joyce defended Senator Hanson's meeting with Robinson earlier this month, a man who has advocated for people to "make war" on Islam and has been convicted of assault on more than one occasion.
"I don't support so much what Tommy Robinson does, but I think it's incredibly important that we understand the social dynamic and how that came about," Mr Joyce said.
"Tommy Robinson only exists because of the fractious nature of England has arrived at, and to deny that is to deny the facts.
"There is a social change, a change in many areas towards a form of Islamisation of the communities, and this has brought real friction and real heat, and has brought about the rise of groups such as Tommy Robinson."
Senator Hanson, who has been spotted holidaying with billionaire donor Gina Rinehart in Italy, is due to speak at the conservative CPAC UK conference in London later this week.
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