After the establishment of the first quarantine station in the Republic of Ragusa (modern-day Dubrovnik) in 1377, the states and principalities of Italy developed a sophisticated system of defensive quarantine in an attempt to protect themselves from the ravages of plague. Using largely unknown and unseen historical maps, this paper reconstructs the extent and operation of the system used. It is shown that a cordon sanitaire existed around the coast of Italy for several centuries, consisting of three elements: (i) an outer defensive ring of armed sailing boats in the Mediterranean and the Adriatic, (ii) a middle coastal ring of forts and observation towers, and (iii) an inner defensive ring of land-based cavalry. The principles established, although not especially successful at the time against a disease of (then) unknown aetiology, are still used today in attempts to control the spread of infections of animal and human populations.