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This work deals with the synthesis of zinc oxide nanoparticles/activated carbon (ZnO NPs/AC) nanocomposites with different weight ratios (3:1, 1:1, and 1:3), where the antimicrobial, antiviral, and cytotoxicity impact of the formulated nanocomposites were evaluated versus the crude ZnO and AC samples. The formula (3:1; designated Z3C1) exhibited the utmost bactericidal effect against Gram positive group, unicellular and filamentous fungi. Regarding Gram negative group, the sample (Z3C1) was remarkably effective against Klebsiella pneumonia, unlike the case of Escherichia coli. Moreover, the whole samples showed negligible cytotoxicity against the human WI38 cell line, where the most brutality (4%) was exerted by 1000 µg/mL of the formula (Z1C3). Whilst, the formula (Z3C1) exerted the apical inhibition impact against Herpes simplex (HSV1) virus. Consequently, the synthesized (Z3C1) nanocomposite was sorted out to be fully characterized via different physicochemical techniques including FTIR, XRD, SEM, TEM, Zeta potential, TGA, and BET. XRD indicated a predominance of the crystalline pattern of ZnO NPs over the amorphous AC, while the FTIR chart confirmed an immense combination between the ZnO NPs and AC. SEM, TEM, and size distribution images illustrated that the fabricated ZnO NPs/AC was in the nanoscale size swung from 30 to 70 nm. The distinctive surface area of composite material, recording 66.27 m 2 /g, clearly disclosed its bioactivity toward different bacterial, fungal, and virus species., (© 2022. The Author(s).)