To date, micropollutants from anthropogenic sources cannot be completely removed from effluents of wastewater treatment plants and therefore enter freshwater systems, where they may impose adverse effects on aquatic organisms, for example, on fish. Advanced treatment such as ozonation aims to reduce micropollutants in wastewater effluents and, thus, to mitigate adverse effects on the environment. To investigate the impact and efficiency of ozonation, four different water types were tested: ozonated wastewater (before and after biological treatment), conventionally-treated wastewater, and water from a river (River Ruhr, Germany) upstream of the wastewater treatment plant effluent. Zebrafish (Danio rerio) embryos were used to study lethal and sublethal effects in a modified fish early life-stage test. Mortality occurred during exposure in the water samples from the wastewater treatment plant and the river in the first 24 h post-fertilization, ranging from 12% (conventional wastewater) to 40% (river water). Regarding sublethal endpoints, effects compared to the negative control resulted in significantly higher heart rates (ozonated wastewater), and significantly reduced swimming activity (highly significant in ozonated wastewater and ozone reactor water, significant in only the last time interval in river water). Moreover, the respiration rates were highly increased in both ozonated wastewater samples in comparison to the negative control. Significant differences between the ozonated wastewater samples occurred in the embryonic behavior and heart rates, emphasizing the importance of subsequent biological treatment of the ozonated wastewater. Only the conventionally-treated wastewater sample did not elicit negative responses in zebrafish, indicating that the discharge of conventional wastewater poses no greater risk to embryonic and larval zebrafish than water from the river Ruhr itself. The sublethal endpoints embryonic- and larval behavior, heart rates, and respiration were found to be the most sensitive endpoints in this fish early life-stage test and can add valuable information on the toxicity of environmental samples.