A reference study on both the nitrogen content in waters and nitrogen and oxygen isotopic signatures characterising nitrate from different sources was conducted within the San River catchment area. Three kinds of catchments were studied: (1) forested and uncultivated; (2) artificially fertilised with nitrate; and (3) fertilised with manure and sewage. Moreover, atmospheric water was studied. The obtained values were found to be similar to others in the literature, with the exception of nitrate from the atmosphere, in regard to which influence reflecting the local conditions was to be noted. The isotopic signature of nitrate in the studied water results from the biogeochemical transformation of nitrogen compounds rather than from the mixing of different sources. The obtained results were statistically distinct and can be used as end-member values in further modelling studies connected with the management of nitrate in river waters, especially under middle-eastern European conditions.