The Neogene extensional province of southern Tuscany in central Italy provides an outstanding example of fossil and active structurally controlled fluid flow and epithermal ore mineralization associated with post-orogenic silicic magmatism. Characterization of the hydrodynamic regime leading to the genesis of the polysulphide deposit (known as Filone di Boccheggiano) hosted within the damage zone of the Boccheggiano Fault is a key target to assess modes of fossil hydrothermal fluid circulation in the region and, more generally, to provide inferences on fault-controlled hydrothermal fluid flow in extensional settings. We provide a detailed description of the fault zone architecture and alteration/mineralization associated with the Boccheggiano ore deposit and report the results of fluid inclusion and stable oxygen isotope studies. This investigation shows that the Boccheggiano ore consists of an adularia/illite-type epithermal deposit and that sulphide ore deposition was controlled by channelling of hydrothermal fluids of dominantly meteoric origin within the highly anisotropic permeability structure of the Boccheggiano Fault. The low permeability structure of the fault core compartmentalized the fluid outflow preventing substantial cross-fault flow, with focused fluid flow occurring at the hangingwall of the fault controlled by fracture permeability. Fluid inclusion characteristics indicate that ore minerals were deposited between 280° and 350°C in the upper levels of the brittle extending crust (lithostatic pressure in the order of 0.1 GPa). Abundant vapour-rich inclusions in ore-stage quartz are consistent with fluid immiscibility and boiling, and quartz ore vein textures suggest that mineralization in the Boccheggiano ore deposit occurred during cyclic fluid flow in a deformation regime regulated by transient and fluctuating fluid pressure conditions. Results from this study (i) predict a strongly anisotropic permeability structure of the fault damage zone during crustal extension, and (ii) indicate the rate of secondary (structural) permeability creation and maintenance by active deformation in the hangingwall of extensional faults as the major factor leading to effective hydraulic transmissivity in extensional terranes. These features intimately link ore-grade mineralization in extensional settings to telescoping of hydrothermal flow along the hangingwall block(s) of major extensional fault zones.