Ozonation is increasingly integrated into wastewater treatment trains to abate micropollutants, which are known to induce adverse effects to aquatic organisms in receiving water bodies.  While ozonation has been found to enhance the biological quality of wastewater as reflected by the reduced toxicity in a range of bioassays, concerns remain with regard to the potential formation of toxic (mutagenic/genotoxic) ozonation byproducts (OBPs) and ozonation transformation products (OTPs) from ozone reactions with the matrix components and with micropollutants, respectively. While most of the toxicity is thought to originate from OBPs as they are typically formed at orders of magnitude higher levels than OTPs, the latter can be used as a tracer to elucidate unknown reaction mechanisms. Except for some specific byproducts such as bromate and nitrosamines, little is so far known about the identity of toxic OBPs/OTPs and their formation pathways. Such an understanding is essential to ensure the optimization of wastewater treatment processes and for the potential implementation of source control measures limiting the introduction of problematic precursors into wastewaters. However, the complexity of wastewater samples along with the typical occurrence of OBPs and OTPs at trace concentration levels constitute main challenges when the identification of unknown reaction pathways is sought. Non-target analysis based on high-resolution mass spectrometry (HRMS) applied synergistically with toxicological assays can constitute a powerful tool to address these challenges and discover unknown toxicologically relevant byproducts. The present study aimed at the identification of unknown reactions that might be relevant with regard to the induction of mutagenicity in ozonated wastewaters. Samples were collected at different wastewater treatment steps: secondary treatment, ozonation, and biological filtration. Solid-phase extraction was performed for enrichment followed by liquid chromatography HRMS/MS and toxicological assays. Prioritization of signals was achieved by comparing their occurrence and intensity in the categorized samples (secondary treatment vs. ozonation vs. biological filtration). Compound identification efforts focused on prioritized compounds classified as OTPs/OBPs and were based on isotope patterns, retention time, and fragmentation spectra. The applied workflow revealed the formation of nitration products during ozonation. Considering the potential toxicity of nitro compounds in general and the recently discovered role of nitrite in the formation of mutagenic compounds during ozonation, this nitration pathway is considered toxicologically relevant. This study highlights how a synergistic approach involving the application of robust non-target screening and toxicological assays can contribute to enhancing our understanding of oxidative wastewater treatment.