(Eco)toxicological assessment of engineered nanoparticles has largely restricted to laboratory conditions, and contribution to the environmental matrix mostly ignored. Thus, the lack of reflection of the environmental matrix makes it harder to evaluate the ecotoxicity of nanoparticles and its mechanism. Therefore, sample pretreatment has a vital role for appropriate (eco)toxicological assessment and reflection of the environmental matrix. The present study aims to investigate the effect of different sample pretreatment strategies on the (eco)toxicity of ZnO nanoparticles and its mechanism. For this purpose, three sample pretreatments and related bacterial bioassay procedure was examined. Moreover, the impact of the sample pretreatment on the toxicity mechanism was investigated using the physicochemical parameters of ZnO nanoparticles and antioxidant responses of bacteria. The results showed that the sample pretreatment affected the physicochemical properties of ZnO nanoparticles, inhibition level and inhibition pathway.