Isabelle Clark was born in Dalnavert in Inverness-shire, Scotland, to parents Alexander and Margaret Clark. She met her husband to be, her half-cousin John Alexander Macdonald, while he was in Great Britain in 1842. The visit included a trip to Douglas on the Isle of Man to see his cousin Margaret Greene née Clark who lived in a small farmhouse near Douglas together with her sister Isabella. Macdonald took an immediate liking to Isabella, suggesting she should visit Kingston the following year, ostensibly to see another of her sisters, Maria, who had married John Alexander Macpherson. Isabella complied, sailing to Canada in 1843 to visit the Macphersons in Kingston, Ontario, as well as the Macdonalds. It was on this trip that after a few months courting, they were engaged and finally married on September 1, 1843, in St. Andrews Church, Kingston. The couple were related by sharing their maternal grandmother; cousin marriage was not uncommon at the time. They lived together in Kingston. Although the marriage got off to a good start, Isabella realized her husband's main interests were Business and politics.

Isabella bore two sons with John. The first was named John, born in New York in August 1847. She was 39 years old. Isabella's health after the birth was not good - it took several months of rest before she was able to return to Kingston. The baby passed away at the age of 13 months. She gave birth to their second son at the age of 41. This child was Hugh John Macdonald, who grew up to be a Lawyer and Politician.

She never recovered her health, and died on December 28, 1857, aged 48. She is buried at the Cataraqui Cemetery, in the Macdonald family plot.