Nitrogen (N) isotopes of aquatic organisms offer a way of differentiating sources of dissolved nitrate species in water. Water quality in the Hartbeespoort Reservoir has been a problem for many decades, causing excessive growth of algae and water hyacinth, both of which further cause human health issues, degradation of environmental water quality, and recreational hazards. Six boreholes and four surface water locations were sampled and analysed for certain water quality parameters and stable water isotopes (H and O). Electrical conductivity and pH were acceptable, but faecal coliforms and Escherichia coli were high in the Crocodile River. δD and δ18O showed that there is little groundwater input to the reservoir and the surface water experiences significant evaporation. Six samples of water hyacinth were analysed for C and N stable isotopes. The δ15N values ranged from 20‰ to 33‰, indicating sewage or manure as the primary source of dissolved N in Hartbeespoort Reservoir. As high dissolved N concentrations cause water hyacinth growth to outstrip any manual, chemical or biological control measures, it is suggested that efforts to control the water hyacinth infestation on Hartbeespoort Reservoir focus on informal settlement sanitation and upgrades to sewage treatment works in the Crocodile River catchment.