During a engagement to Lichfield Cathedral on the start of the week, the King was heckled by a man who asked him about his brother's relationship with disgraced financier Jeffrey Epstein.
The man asked how long the King had been aware about his brother's association with Epstein, and also questioned whether he had instructed the police to "cover up" for Andrew.
Despite the protest, there were throngs of fans and applause for the King, who was visiting the region for an ceremony.
The Duke of York's links with Epstein have been under close examination in recent weeks. The royal, who relinquished his royal duties earlier this month, has repeatedly denied any misconduct.
"How long have you known about Prince Andrew and Jeffrey Epstein? Did you request the authorities to cover up for Andrew?"
The monarch did not respond but continued greeting supporters, some of whom chanted "Long live the King". One fan can be overheard requesting him for a embrace.
A female bystander outside the venue was also heard telling the protester to "leave" and to "be quiet".
The scandal over Prince Andrew's connections to Jeffrey Epstein has been reignited by misconduct claims detailed in Virginia Giuffre's posthumously-published book.
In the memoir, the accuser alleges she had sexual encounters with the prince on three separate occasions, including once with Jeffrey Epstein and "several other minors"
Prince Andrew, who agreed to a out-of-court agreement with the plaintiff in 2022, has repeatedly denied any misconduct. In 2019, he told a news program he did not remember meeting Ms Giuffre "ever", and that they "did not have any sort of intimate relations."
But in recent times, new inquiries have been asked about how he is able to fund his lifestyle despite not being a active member of the royal family.
The revelation that he has only ever paid a token annual rent on his Royal Lodge has intensified the examination over his living arrangements - even though he made significant contributions up front, including for improvements.
The Liberal Democrats said he should be summoned before MPs to give testimony about his rental agreement of his lodge.
But last Thursday, the government declined to give parliamentarians time to debate the prince's titles or his home, despite increasing calls for investigation.
Buckingham Palace has not commented on whether the Duke of York might move out of his home - or where he might reside.