Antoinette of Bourbon was born on 25 December 1494 at the Chateau de Ham, in the Somme department, Picardy, France.

She exhibited considerable administrative talent at domestic economy as well as in the running of the vast Guise dominions surrounding their chateau of Joinville.

Antoinette exerted a powerful influence on the childhood of her granddaughter Mary, Queen of Scots, during the latter's thirteen-year sojourn in France, and was one of her principal advisors.

Antoinette acted as proxy for her daughter, Mary of Guise, during the betrothal ceremony of the Queen of Scots and the Dauphin Francis on 19 April 1558.

[4] Her son Francis, Duke of Guise was held to be responsible for the anti-Protestant Massacre of Wassy on 1 March 1562 which was one of the early atrocities in the French Wars of Religion.