Identifying the origin of nitrate is important for the control and management of groundwater quality in aquifer systems. In the southern Apennines (Italy), the Mount Vulture volcanic aquifer is a large and valuable resource of potable and mineral water supply. Unfortunately, signs of anthropogenic impact, especially nitrogen contamination, have recently become evident. In this study, and for the first time, stable isotope ratios (δ15N and δ18O) of NO3 − were determined in groundwater to identify their origins and evaluate the presence of transformation processes. The Mount Vulture groundwaters are meteoric in origin, as demonstrated by measurements of δD and δ18O, and can be divided into two distinct areas based on their NO3 − content. In the southeastern area, characterized by active agricultural land use, the high NO3 − content and the δ15N–NO3 isotopic values are due to anthropogenic contamination (inorganic fertilizer). In groundwaters from the western area, the NO3 − contents below 4 mg/L and the δ15N–NO3 values can be associated at organic soil N. Evidence for local denitrification may be assumed in a few groundwater samples of the western area showing relatively heavy δ15N values and low concentrations of nitrate. Finally, the low measured δ18O values indicate that nitrification occurred in both investigated areas.