Although conventional wastewater treatment technologies are effective at removing many contaminants of emerging concern (CECs) from municipal wastewater, some contaminants are not removed efficiently. Ozonation may be a treatment option for reducing the concentrations of recalcitrant CECs in wastewater, but this process may generate toxic transformation products. In the present study, we conducted semibatch experiments to ozonate municipal wastewater effluent spiked with 5 commonly detected CECs. The purpose of the present study was to evaluate whether ozonation increased or decreased biological responses indicative of sublethal toxicity in juvenile rainbow trout (Oncorhynchus mykiss) injected intraperitoneally (i.p.) with extracts prepared from ozonated and nonozonated wastewater effluent. Blood, liver, and brain tissues were collected from the fish at 72 h post injection for analysis of a battery of biomarkers. In fish i.p. injected with the extracts from nonozonated wastewater effluent, significant induction of plasma vitellogenin (VTG) was observed, but ozonation of the municipal wastewater effluent spiked with CECs significantly reduced this estrogenic response. However, in fish injected with extracts from spiked municipal wastewater effluent after ozonation, the balance of hepatic glutathione in its oxidized (glutathione disulfide [GSSG]) form was altered, indicating oxidative stress. Levels of the neurotransmitter serotonin were significantly elevated in brain tissue from trout injected with the extracts from ozonated spiked municipal wastewater effluent, a biological response that has not been previously reported in fish. Other in vivo biomarkers showed no significant changes across treatments. These results indicate that ozonation reduces the estrogenicity of wastewater, but may increase other sublethal responses. The increase in biomarker responses after ozonation may be because of the formation of biologically active products of transformation of CECs, but further work is needed to confirm this conclusion. Environ Toxicol Chem 2018;37:274-284. C 2017 SETAC