The removal of contaminants of emerging concern from urban wastewater treatment plants (WWTPs) remains a challenge to promote safe wastewater reuse practices. Macrolides are the most abundant antibiotics detected in untreated wastewater and their concentration in WWTPs effluents is only partially reduced by conventional treatments. Among several advanced oxidation processes (AOPs), photocatalysis has demonstrated the capability to effectively remove pharmaceuticals from different aqueous matrices. Recently, ZnO has emerged as an efficient, promising, and less expensive alternative to TiO2, due to its photocatalytic capability and attitude to exploit better the solar spectrum than TiO2. In this study, the behaviors of ZnO photocatalysis were evaluated using a representative macrolide antibiotic, spiramycin (SPY), in aqueous solutions and urban wastewater. After 80 min of photocatalysis, 95–99% removal of SPY was achieved at 1 g L−1 ZnO concentrations in aqueous solutions and wastewater, respectively. After treatment, the effluent toxicity, evaluated using the bacterium Aliivibrio fischeri, the green alga Raphidocelis subcapitata, and the crustacean Daphnia magna ranged between slight acute and high acute hazard. Filterable and ultrafilterable Zn concentrations were quantified in treated effluents and shown to be high enough to contribute to the observed toxicity.