During a visit to Lichfield Cathedral on Monday, the King was confronted by a man who questioned him about his brother's connections with convicted sex offender Jeffrey Epstein.
The individual asked how long the monarch had been known about his brother's relationship with Epstein, and also raised doubts about whether he had asked the police to "hide" for Andrew.
Despite the protest, there were crowds of well-wishers and applause for the monarch, who was visiting the region for an engagement.
The Duke of York's connections with Epstein have been under renewed focus in the past few weeks. The prince, who gave up his titles earlier this month, has strongly denied any wrongdoing.
"For how long have you known about Andrew and Jeffrey Epstein? Did you request the authorities to conceal for him?"
The monarch did not respond but carried on greeting fans, some of whom chanted "Long live the King". One supporter can be overheard requesting him for a hug.
Another woman outside the venue was also heard telling the heckler to "go away" and to "shut up".
The scandal over the Duke of York's links to Jeffrey Epstein has been brought back into focus by sexual abuse allegations detailed in Ms. Giuffre's recently released memoir.
In the memoir, Ms Giuffre alleges she had sexual encounters with the prince on three separate occasions, including once with Epstein and "several other minors"
Prince Andrew, who agreed to a out-of-court agreement with Ms Giuffre in 2022, has consistently denied any wrongdoing. In 2019, he told a news program he did not recall meeting Ms Giuffre "at all", and that they "never had any sort of intimate relations."
But recently, fresh questions have been raised about how he is able to finance his way of life despite not being a active member of the royal family.
The disclosure that he has only ever paid a token annual rent on his Royal Lodge has intensified the scrutiny over his living arrangements - even though he made significant contributions up front, including for improvements.
The political party said he should be called in front of members of parliament to give evidence about his lease of his lodge.
But last week, the government refused to give MPs time to discuss the prince's status or his residence, despite increasing calls for scrutiny.
The royal household has not commented on whether the Duke of York might relocate of his current residence - or where he might reside.
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