Advanced wastewater treatment technologies are generally known to be an effective tool for reducing micropollutant discharge into the aquatic environment. Nevertheless, some processes such as ozonation result in stable transformation products with often unknown toxicity. In the present study, whole effluents originating from nine different steps of advanced treatment combinations were compared for their aquatic toxicity. Assessed endpoints were survival, growth and reproduction of Lumbriculus variegatus, Daphnia magna and Lemna minor chronically exposed in on-site flow-through tests based on standard guidelines. The treatment combinations were activated sludge treatment followed by ozonation with subsequent filtration by granular activated carbon or biofilters and membrane bioreactor treatment of raw wastewater followed by ozonation. Additionally, the impact of treated wastewater on the immune response of invertebrates was investigated by challenging D. magna with a bacterial endoparasite. Conventionally treated wastewater reduced reproduction of L. variegatus by up to 46%, but did not affect D. magna and L. minor with regard to survival, growth, reproduction and parasite resistance. Instead, parasite susceptibility was significantly reduced in D. magna exposed to conventionally treated as well as ozonated wastewater in comparison to D. magna exposed to the medium control. None of the three test organisms provided clear evidence that wastewater ozonation leads to increased aquatic toxicity. Rather than to the presence of toxic transformation products, the affected performance of L. variegatus could be linked to elevated concentrations of ammonium and nitrite that likely resulted from treatment failures.