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In this study, a sustainable and eco-friendly method was used to prepare zinc oxide nanoparticles (ZnO-NPs) using a sea cucumber aqueous extract. Then, ZnO-NPs were characterized by instrumental analysis (UV-vis, HR-TEM, XRD, FT-IR, and DLS) and evaluated for their possible antibacterial, antifungal, and insecticidal activities. Additionally, the toxicity of ZnO-NPs was evaluated <i>in vivo</i> against Nile Tilapia (<i>Oreochromis niloticus</i>). The sea cucumber was collected from the Gulf of Suez (Red Sea) at Al-Ain Al-Sokhna coast in Egypt and identified as <i>Holothuria impatiens</i>. The prepared <i>Hi</i>-ZnO-NPs peaked at 350 nm in UV–Vis spectral analysis. They showed quasi-spherical shaped particles with sizes ranging from 13 nm to 47 nm and a predominate size of 26 nm as indicated by HR-TEM. The XRD pattern of <i>Hi</i>-ZnO-NPs revealed a crystalline phase with an average size of 17.2 nm as calculated by Debye–Scherrer equation. FTIR analysis revealed the possible role of <i>H. impatiens</i> biological molecules in the biosynthesis process of ZnO-NPs. <i>Hi</i>-ZnO-NPs showed a negative zeta potential of −19.6 mV, demonstrating moderate stability. Biosynthesized <i>Hi-</i>ZnO-NPs revealed broad antimicrobial activity against Gram-positive bacteria (<i>S. aureus</i> ATCC 25923 and <i>E. feacalis</i>), Gram-negative bacteria (<i>S. typhi</i>, <i>K. pneumonia</i> and <i>E. coli</i>), and filamentous fungi (<i>Aspergillus niger</i>). <i>Hi</i>-ZnO-NPs demonstrated larvicidal activity against the mosquito, <i>Culex pipiens</i> (LC₅₀ = 2.756 ppm and LC₉₀ = 9.294 ppm), and adulticidal action against the housefly, <i>Musca domestica</i> (LD₅₀ = 4.285 ppm and LD₉₀ = 22.847 ppm). Interestingly, <i>Hi</i>-ZnO-NPs did not show mortality effects against <i>Nile tilapia</i> fish (<i>Oreochromis niloticus</i>), highlighting the potential safety of <i>Hi</i>-ZnO-NPs to highly exposed, non-target organisms. However, histopathological and hematological investigations provided dose-dependent impacts of <i>Hi</i>-ZnO-NPs to <i>Nile tilapia</i>. Overall, data provide an eco-friendly approach for synthesizing novel <i>Hi</i>-ZnO-NPs with multiple biomedical properties and potentially low toxicity to <i>Nile tilapia</i> fish.