Born in Vienna on 30 April 1539,[1] Barbara was the eleventh child and eighth daughter of Ferdinand I, Holy Roman Emperor and Anna of Bohemia and Hungary.

[2][3] In the winter of 1547, the widowed Emperor Ferdinand I entrusted all his unmarried daughters to the care of nuns in the monastery in Innsbruck, where Barbara lived until her marriage.

Only once, in 1552, during the invasion of the Tyrol by the Protestant army under the command of Maurice, Elector of Saxony, did Barbara and her sisters Magdalena, Margaret, Helena and Joanna, spend some time outside the monastery at Bruneck Castle.

The characteristic features of her education, based on the writings of the Jesuits Peter Canisius and Diego Laynez, were religiosity and charity.

In 1562, several suitors came to Emperor Ferdinand I to ask for the hand of his youngest daughter Joanna, among them John Sigismund Zápolya, Francesco de' Medici, Crown Prince of Florence and Alfonso II d'Este, Duke of Ferrara, Modena and Reggio.

In November of the same year, she and Joanna arrived in Trento, where Pope Pius IV sent his legates to conduct a double marriage ceremony; however, because of the renewed conflict between the grooms, the brides had to go to the respective capitals (Ferrara and Florence) of their future spouses to be wedded.

When, a year after the wedding, Alfonso II participated in the war against the Ottoman Empire, Barbara was sincerely worried about her husband.

The experience negatively impaired her health; from that time she was reported as being constantly ill.[1] Despite being a devout Catholic, Barbara was able to forge an excellent relationship with her Protestant mother-in-law Renée of France.

In the early one, a work of Giuseppe Arcimboldo, she is depicted during the period of her negotiations of her marriage to Alfonso II in 1563-1564, in a portrait presumably made for her future husband.