Mutagenicity formation by low pressure (LP)-UV/H2O2 treatment of nitrate-rich water containing natural organic matter (NOM) was investigated. Laboratory-grade water samples spiked with either Pony Lake NOM or Suwannee River NOM (4 mg L−1 in both cases) and nitrate (50 mg L−1) were irradiated with UV fluences of 0, 1500 and 2000 mJ cm−2 and a H2O2 dose of 15 mg L−1 and tested for mutagenicity with the Ames II assay. LP-UV photolysis of nitrate in the presence of Pony Lake NOM caused a significant increase in the Ames II assay response and low concentrations of nitrite (0.08–0.09 mg NO2− L−1) and nitrophenols (0.014–0.046 μg L−1) were detected. Suwannee River NOM produced the same nitrite levels but no significant responses in the Ames II assay were observed. Additionally, samples collected from a drinking water treatment plant in the UK using LP-UV/H2O2 treatment were analysed with the Ames II assay. LC-OCD fractionation and SUVA measurements were performed to observe any changes in the properties of NOM. Significant differences in the mutagenicity response were observed between the treatment steps as well as between the two sampling periods. However, with respect to standard thresholds, none of the samples were found to be mutagenic towards the Salmonella typhimurium strain TA98 used.