Nitrates are among the most common groundwater contaminants worldwide, and the same situation is present within the Zagreb aquifer. The Zagreb aquifer presents the only source of potable water for inhabitants of the City of Zagreb and part of Zagreb County. Isotopic composition of water (δ2H and δ18O) and nitrates (δ15N and δ18O), groundwater chemistry, and molar ratios, in combination with correlation and multivariate statistical methods, have been used for the estimation of nitrate origin. Nitrate stable isotopes excluded synthetic fertilizer as the main source of nitrate contamination. They showed insignificant influence of denitrification on nitrate concentrations but could not define the main source of nitrate contamination. The usage of molar ratios, especially NO3−/K+, helped to clarify this issue. Waste water has been defined as the main source of nitrate contamination. All results indicate that nitrogen in a large extent enters the aquifer in the form of ammonium ion, which is transformed to nitrates by the process of nitrification.