Built on a project by Antonio and Giuliano da Sangallo as an enlargement of the square fortalice (known as the "Pisans’ quadrature") erected next to the Mathilde stronghold, the Old Fortress was also the Livorno residence of Cosimo I de’ Medici who, in 1544, ordered the construction of the Palazzotto on the Canaviglia bastion that commanded the entrance to the port. Cosimo, then duke of Florence, also had a famous cistern excavated, the water of which was praised by Francesco Redi.

On April 2, 1662, the tower of the Fortress was the theatre for observations on the motion of projectiles, conducted by Cimento academicians to experimentally confirm the conclusions of Galileo Galilei. Between late 1657 and early 1658, the city of Livorno had also been the scene of several experiments by the Cimento academicians on "congealments", that is to say on the freezing of liquids.

The Fortress was not only a place for the experimental verification of physics, but also a site of "naturalistic observation". In his Guida di Livorno, Piombanti tells of how, in October 1734, a storm had beached a whale at the foot of the Fortress, and of how the event recurred in January of 1753, when two cetaceans were killed near the port with cannon-shots fired from the bastion above.