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Here, we report on the synthesis of zinc oxide nanoparticles (ZnO NPs) and selenium nanowires (Se NWs) using microbial exopolysaccharides (EPS) as a mediator and then examine their antibacterial and ecotoxicity effects in vitro and in vivo, respectively. At 100 µg/mL, EPS, EPS-ZnO NPs, and EPS-Se NWs all exhibited potent in vitro antibacterial properties, drastically inhibiting the development of aquatic Gram^(-) pathogens. In addition, antibiofilm studies using a microscope revealed that EPS, EPS-ZnO NPs, and EPS-Se NWs at 75 µg/mL prevented biofilm development. Furthermore, the in vivo toxicity was carried out via <i>Danio rerio</i> embryos and <i>Ceriodaphnia cornuta</i>. <i>Danio rerio</i> embryos were determined at different time intervals (6 hpf, 12 hpf, 24 hpf and 48 hpf). The maximum survival rate (100%) was obtained in a control group. Correspondingly, EPS, EPS-ZnO NPs and EPS-Se NWs treated embryos showed a considerable survival rate with 93.3%, 86.7% and 77.2%, respectively, at 100 µg/mL for 48 hpf. The total mortality of <i>C. cornuta</i> was seen at 100 µg/mL, with 56.7% in EPS, 60.0% in EPS-ZnO NPs, and 70.0% in EPS-Se NWs. For <i>C. cornuta</i>, the LC₅₀ values for EPS, EPS-ZnO NPs, and EPS-Se NWs were 90.32, 81.99, and 62.99 µg/mL, respectively. Under a microscope, morphological alterations in <i>C. cornuta</i> were analyzed. After 24 h, an amount of dark substance was seen in the guts of <i>C. cornuta</i> exposed to 100 µg/mL, but in the control group, all of the living <i>C. cornuta</i> were swimming as usual. Our results show that EPS and EPS-ZnO NPs were less harmful than EPS-Se NWs, and that they were successfully employed to shield freshwater crustaceans from the toxins in aquatic environments.