Daughter of Ranald Macdonald (d. 1723) and his second wife, Marion. In 1750, she married Allan MacDonald, with whom she had at least two daughters and five sons.
Despite her family's government allegiance, she helped the young pretender, Prince Charles Edward Stuart, escape capture following his final defeat at the Battle of Culloden, leading to her imprisonment in the Tower of London, from which she was released in 1747.
She lived with her husband on Skye's Trotternish peninsula until 1774, when they emigrated to North Carolina in 1774. Her husband was taken prisoner by Revolutionaries at the Battle of Moore's Creek Bridge in 1776, being released 18 months later as part of a prisoner exchange. He became a Captain in the 84th regiment stationed in Nova Scotia, but Flora, being homesick and of poor health, decided to return to Skye in 1779, Allan rejoining her only after the conclusion of the war in 1784. She spent the intervening years living with various members of their family, and also stayed for some time with her daughter Anne and son-in-law, Major-General Alexander Macdonald, at Dunvegan Castle.
Boswell and Dr Johnson stayed with Flora Macdonald and her husband at Kingsburgh on September 12-13, 1773. He described her, in his Journal of a Tour to the Hebrides, as "a little woman, of a mild and genteel appearance, mighty soft and well-bred." Boswell further wrote that they had "as genteel a supper as one would wish to see," and a punch that was "superexcellent".
At night, Johnson slept in the same bed as Prince Charles had done some 27 years before, and Boswell remarked in his journal that "To see Mr Samuel Johnson lying in Prince Charles’s bed, in the Isle of Skye, in the house of Miss Flora Macdonald, struck me with such a group of ideas as it is not easy for words to describe as the mind perceives them."
Boswell later gave an extensive account of the prince's flight following the Battle of Culloden in 1746 and the Macdonalds' part in it in Journal of a Tour to the Hebrides, followed by a discussion of his and Dr Johnson's views on monarchy and the proper right of succession.