Periodate (PI) shows promising potential as an oxidant for wastewater treatment; however, its impact on the toxicity of wastewater remains unknown. Here, we found that with 100 μM PI addition, the cytotoxicity of wastewater increased from 4.8 to 7.6 mg-Phenol/L to 9.5 to 12.8 mg-Phenol/L, and genotoxicity increased from 0.3 to 0.9 μg-4-NQO/L. Interestingly, hypoiodous acid (HOI) was not detected during the reaction, and there was no observed increase in the concentration of total organic iodine (TOI). CHON components in dissolved organic matter changed most obviously in PI oxidation, which might serve as primary precursors for toxic byproducts. Cytotoxicity of typical nitrogen-containing precursors of tryptophan, lysine, phenylalanine, and tyrosine after PI oxidation increased from not detected to 14.7, 2.4, 4.1, and 3.2 mg-Phenol/L, respectively. Here, four nonhalogenated aromatic nitrogenous byproducts (N-DBPs) of 3-hydroxyquinoline, 4-hydroxyquinoline, benzopyridine, and benzopyrrole were confirmed using standards, and four byproducts such as 2-formylbenzonitrile were tentatively proposed. The cytotoxicity of the four confirmed byproducts was comparable to those known N-DBPs such as nitrosamines, suggesting attention should be given to these nonhalogenated but nitrogenous byproducts. The four confirmed byproducts were detected in two PItreated wastewater samples with concentrations of 0.8, 0.98, 0.52, and 0.0038, and 18.28, 1.50, 0.57, and 0.0074 μg/L, respectively, with contributions less than 1.5% to the overall cytotoxicity. Further investigations are warranted to elucidate the primary drivers of toxicity in PI-treated wastewater.