Giulia Ammanati, Galileo's mother, came from a family that had originated from Pescia and settled in Pisa around 1536. Giulia was born in 1538 and had three sisters, Diamante, Dorotea and Ermellina, and one brother, Leone. The only thing we know about her father Cosimo, a lumber merchant, is that on the day his daughter married Vincenzo Galilei (c.1520-1591), on July 5, 1562, he was already deceased. It was Leone who provided the dowery, and in addition to handing over a sum of money he furnished the newlyweds with room and board for one year.

Giulia, with her children, rejoined her husband in Florence in 1574. At the death of Vincenzo, in 1591, the burden of supporting his mother and siblings fell to young Galileo, who had recently become a mathematics instructor at the University of Pisa.

A letter survives from this period, in which Galileo cites an illness of his mother's as the reason for missing his lessons at the university, for which he had to pay a fine. Galileo actually speaks of a "grave, almost mortal infirmity", which actually must not have been all that serious.

Some of Giulia's letters have survived from Galileo's time in Padua, in which she complains of her son's neglect or reminds him of the debt he owes for his sister Virginia's dowery. Now and then she went to Padua, and her difficult character made these visits very hard on young Galileo. In 1604 Silvestro Pagnoni, an employee of Galileo's, reported him to the Inquisition for practicing judicial astrology and for non-observance of his religious duties. Among these accusations he also cites the testimony of Giulia Ammannati about her son: "I am well aware as his mother that he never goes to confession or takes holy communion". She even had him spied on to ascertain if he was going to mass or to his girlfriend's, Marina Gamba.

In the latter half of 1609, after having paid him a visit in Padua, Giulia returned to Florence, bringing along her granddaughter Virginia, Galileo first-born child, whom she took care of until Galileo's return to Tuscany the following year.

Giulia Ammannati died in August of 1620 and was buried in the Church of Carmine in Florence.