On 18 December 1565, he married Joanna of Austria, youngest daughter of Holy Roman Emperor Ferdinand I and his wife Anne of Bohemia and Hungary, after Princess Elizabeth of Sweden, among others, had been considered. By all reports, it was not a happy marriage. Joanna was homesick for her native Austria, and Francesco was neither charming nor faithful. Joanna died at the age of thirty-one in 1578.
Soon after Grand Duchess Joanna had died, Francesco went on to marry his Venetian mistress, Bianca Cappello, after aptly disposing of her husband, a Florentine bureaucrat. Because of the quick remarriage and similar occurrences among the Medici (Francesco's younger brother Pietro had reportedly killed his wife), rumours spread that Francesco and Bianca had conspired to poison Joanna. Francesco reportedly built and decorated the Villa di Pratolino for Bianca. She was, however, not always popular among Florentines. They had no legitimate children, but Bianca had borne him a son, Antonio (29 August 1576 – 2 May 1621), in his first wife's lifetime. Following the death of Francesco's legitimate son Filippo in 1582, Antonio was proclaimed heir. Francesco also adopted Bianca's daughter by her first marriage, Pellegrina (1564- ?).
Like his father, Francesco was often despotic, but while Cosimo had known how to maintain Florentine independence, Francesco acted more like a vassal of the Habsburgs of Austria and Spain. He continued the heavy taxation of his subjects to pay large sums to the empire.
He had an avid interest in manufacturing and sciences. He founded porcelain and stoneware manufacture, but these did not thrive until after his death. He continued his father's patronage of the arts, supporting artists and building the Medici Theater as well as founding the Accademia della Crusca. He was also passionately interested in chemistry and alchemy and spent many hours in his private laboratory and curio collection, the Studiolo in the Palazzo Vecchio, which held his collections of natural items and stones and allowed him to dabble in chemistry and alchemical schemes.
Francesco and Bianca died on 19 and 20 October, both at the Medici Villa in Poggio a Caiano. Although the original death certificates mention malaria, it has been widely speculated that the couple was poisoned, possibly by Francesco's brother Ferdinando. While some early forensic research supported the latter theory, forensic evidence from a study in 2010 found the parasite Plasmodium falciparum, which causes malaria, in the skeletal remains of Francesco I, which strongly bolstered the infection theory and the credibility of the official documents. Francesco was succeeded by his younger brother Ferdinando.
In 1857, all members of the Medici family were exhumed and reburied in the place where they still lie today. The painter Giuseppe Moricci attended the ceremony and depicted Francesco with a facial droop, a right claw hand appearance, the right shoulder internally rotated, the right calf muscle wasted and a right clubfoot confirmed by orthopaedic footwear within the coffin. These are the signs of a right-sided stroke possibly within the internal capsule. The presence of the orthopaedic footwear suggests that this stroke happened significantly before his death. During life, in his official portraits, the grand duke was always depicted as being in perfect physical condition. The cause of his stroke is not known, but malaria is known to cause this condition
There is a famous portrait of Francesco as a child by Bronzino that hangs in the Uffizi Gallery in Florence. Francesco's marriage to Bianca and the couple's death was exploited by Thomas Middleton for his tragedy Women Beware Women, published in 1658.
Francesco and Joanna had seven children: