The girlfriend of Prince William was pictured stalking with the Prince of Wales on the 50,000-acre estate in Aberdeenshire.
But the League Against Cruel Sports voiced concerns after the photographs appeared in a number of newspapers.
"I don't think the Royal Family does itself any good whatsoever by doing this," said Barry Hugill of the League.
"Charles and his son have an appalling record of slaughtering animals for fun and it's terribly sad that William is introducing his girlfriend to such a barbaric and unacceptable pastime."
The pictures have fuelled speculation that Miss Middleton may soon become engaged to Prince William. They show her wearing a camouflage jacket and dark jeans, with gaiters over her boots. Charles, dressed smartly in a tie, appeared to be instructing her.
Miss Middleton, 25, is spending a long weekend with Prince William, but reports say William did not go on the stalking trip.
Prince William and Miss Middleton are staying at Birkhall, Charles's home on the estate, and are guests of the Prince and the Duchess of Cornwall.
The Queen left Balmoral Castle last week to return to Buckingham Palace after her summer holiday there.