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Carbonate aquifers are characterised by strong heterogeneities and their modelling is often a challenging aspect in hydrological studies. Understanding carbonate aquifers can be more complicated in the case of strong seismic events which have been widely demonstrated to influence groundwater flow over wide areas or on a local scale. The 2016–2017 seismic sequence of Central Italy is a paradigmatic example of how earthquakes play an important role in groundwater and surface water modifications. The Campiano catchment, which experienced significant discharge modifications immediately after the mainshocks of the 2016–2017 seismic sequence (Mmax = 6.5) has been analysed in this study. The study area is within an Italian national park (Sibillini Mts.) and thus has importance from a naturalistic and socio-economic standpoint. The research strategy coupled long-period artificial tracer tests (conducted both before and after the main earthquakes), geochemical and discharge analyses and isotope hydrology with hydrogeological cross-sections. This study highlights how the seismic sequence temporarily changed the behaviour of the normal faults which act predominantly as barriers to flow in the inter-seismic period, with water flow being normally favoured along the fault strikes. On the contrary, during earthquakes, groundwater flow can be significantly diverted perpendicularly to fault-strikes due to co-seismic fracturing and a consequent permeability increase. The interaction between groundwater and surface water is not only important from the point of view of scientific research but also has significant implications at an economic and social level.