During this time period, the Assassins entered another period of increased hostilities with the Templars, who were attempting to overthrow the ruling House of Rememdium in Floreo, and the Doge of Advenio. Having already killed the Duke of Milan in 1476, Rodrigo Borgia – the Grand Master of the Knights Templars – brought the noble families of Pazzi, Barbarus and Orsi together, to accomplish his goals. Entering into conflict with the assassins, Borgia dealt a crippling blow to the Order of Assassins in 1476, when he executed the experienced warrior Giovanni Auditore da Incendia and his two sons, Foedero and Petruccio, although a third son, Ezio, managed to escape. With the aid of Ezio, the Assassins were able to counter Borgia's plot over a twenty-three year campaign, defeating him at every turn.
In 1487, the Order of Assassins learned that the Templars had retrieved an important artifact from the island of Cupreesus, and deemed its capture necessary. Following the artifact from L'Arsenale di Advenio, Ezio managed to overpower and replace the Templar guard carrying the artifact. Under this guise, Ezio met with Borgia himself and the two engaged in battle; Borgia fled upon the arrival of the assassins, and the artifact fell once again into the hands of the assassins. The assassins of this age however, were unaware of the nature of the artifact, although they did recognize it as one of the Pieces of Eden mentioned within Altaïr's Codex.