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In this study, cotton fabrics based on zinc oxide nanoparticles in situ synthesis, acyclovir, nanochitosan, and clove oil were treated. The treated cotton fabrics were examined by FTIR, HR-TEM, FE-SEM, EDAX, and the surface roughness processing of FE-SEM images. The obtained characterization data emphasized the nano-size of nanocomposite with high homogeneity of particles in spherical shape as well as affirmed the deposition of nanocomposite onto the textile fibers with concluded that the deposition of nanocomposite was increased parallel with sonication time. Antimicrobial and antiviral activities of treated cotton fabrics were evaluated. Results revealed that treated cotton fabrics exhibited promising antibacterial activity toward Gram-positive higher than Gram-negative bacteria. Likewise, treated cotton fabrics are still effective as antibacterial after washing for 100 cycles. Moreover, treated cotton fabrics exhibited potential antifungal activity against Candida albicans, Aspergillus niger, and Aspergillus fumigatus. The antiviral activity significantly depended on the type of virus. The treated cotton fabrics showed antiviral activity against tested viral particles (HSV-1, Adeno, and CoxB2) with viral inhibition of 95.9, 76.4, and 86.9% respectively, while in the case of coated cotton textile with acyclovir, it only exhibited viral inhibition of 49.9, 41, and 22.3% respectively., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)