A multi-layered aquifer system (eastern Po plain, northern Italy) was investigated by means of isotopic data, with the goal of quantifying groundwater recharge from different sources and assessing the intrinsic vulnerability of aquifers to surface sources of contamination. The geology of the area is based on a stratigraphic alternation of several sandy aquifers and silty–clayey aquitards, down to a maximum depth of 200 m b.g.s. Water isotopes (δ18O, δ2H), and hydrochemical analyses were performed on groundwater samples collected from a regional network of 70 boreholes distributed on an area of almost 2000 km2. In addition to the regional sampling, detailed vertical isotopic profiling was performed in one location by means of groundwater and sediment samples collected through the whole sequence of aquifers and aquitards. Water isotopes indicated mixing from different sources of recharge (i.e., vertical recharge, Po river, deeper aquifers). Mixing calculations were used to quantify the contributions to the aquifers from the different sources. The vertical profiling allowed for integrating and validating the interpretations at a regional scale. The recharge pattern defined for the different aquifers was translated into an index of hydrogeologic interconnections with the surface, which represents a physically based proxy of the intrinsic vulnerability of the aquifers to surficial sources of contamination. The investigated setting can be considered to be representative of many other anthropized and groundwater demanding plain settings around the world. Thus, the proposed method represents a valuable approach for such settings both for recharge quantification (e.g., to be used as input for numerical modeling) and for a physically based assessment of the intrinsic vulnerability.