A multi-isotope approach in combination with hydrochemical data and borehole logging is applied to identify the source of brackish groundwater in a borehole in the well field of Neksø Municipal Waterworks in Bornholm, Denmark. The aquifer lithology consists of fractured Lower Cambrian sandstones resting on Proterozoic crystalline basement. The water body in the studied borehole is significantly stratified with respect to the hydrochemical and isotopic signatures and reveal a Na–Ca–Cl–HCO3 water type and TDS values >1,000 mg/L below a halocline at 40–55 m below measurement point (bmp). The occurrence of brackish groundwater is remarkable for this aquifer, which otherwise yields potable groundwater of good quality. The stable isotope (18O and 2H) compositions indicate a meteoric origin of the brackish groundwater, which rules out seawater intrusion into the aquifer. 14C activities show apparent 14C ages of the brackish groundwater in the range 2200–4300 yr (BP), whereas the freshwater samples above the halocline indicate modern age. Hydrochemical (Cl/Br and Sr) and isotopic studies (18O, 2H and 87Sr/86Sr) of the brackish groundwater point to a well-mixed and homogeneous water body reflecting long water–rock interaction and suggest a contribution of palaeowater from the fractured crystalline basement which has intruded into the Lower Cambrian sandstone aquifer.