Two zealots from Animal Rising defaced the 8ft by 6ft painting in protest at his role as patron of the RSPCA, at the centre of cruelty allegations.
One with a manbun was filmed pasting over Charles’ face with the grinning mug of Wallace, the hapless cheese-loving owner of Gromit the dog in the TV animations.
A second activist stuck on a speech bubble, which read: “No cheese, Gromit. Look at all this cruelty on RSPCA farms!”
Queen Camilla once dubbed Wallace and Gromit her husband’s “favourite people in the world”.
After Charles succeeded to the throne, their creator Nick Park produced a painting captioned: “Cracking Coronation Your Majs!”
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Serial eco-protesters Daniel Juniper and Ben Thomas struck at noon at the Philip Mould Gallery in London’s Pall Mall near Buckingham Palace.
Unveiled last month by artist Jonathan Yeo, the canvas is protected by a screen, and the stickers were easily removed.
At the weekend, Animal Rising, an offshoot of Extinction Rebellion, alleged animal cruelty in 45 farms covered by the RSPCA Assured guarantee on welfare standards.
Juniper said: “With King Charles being such a big fan of Wallace and Gromit, we couldn’t think of a better way to draw his attention to the horrific scenes.”
The campaigners claim 280 legal breaches and 94 of Defra regulations were found on the RSPCA Assured farms.
The charity insisted it took welfare concerns “extremely seriously” and was investigating.
But a spokesperson said: “We are shocked by this vandalism of His Majesty King Charles, our Patron’s, portrait.
“We welcome scrutiny of our work but we cannot condone illegal activity of any kind. Our staff and volunteers work extremely hard rescuing, caring for, and speaking up for animals.”
Gallery owner Philip Mould said security was being reviewed. He added: “No damage was done. The stickers only remained up for ten or 15 seconds and then were taken down by staff. I asked the individuals to leave and they did.”
The Met added: “The gallery did not wish to report a crime and, as such, there is no further action by police.”
Buckingham Palace declined to comment.