Abstract Antimicrobial coatings offer a promising solution for enhancing the efficacy of materials used to fabricate protective equipment for healthcare personnel. Given the rapid spread of respiratory diseases caused by pathogenic bioaerosols, our study delves into probing the antimicrobial properties of a sputtered ZnO nanolayer deposited onto polypropylene fabrics earmarked for the production of respiratory protective gear such as facemasks. A comprehensive methodology was developed to assess the immediate antimicrobial effect of the zinc oxide nanolayer against bioaerosols laden with four DNA or RNA viral surrogates and eight aerobic and anaerobic bacterial species. Additionally, its antimicrobial efficacy was measured over time across contact durations ranging from 0.5 to 24 h. The ZnO nanolayer exhibited an immediate reduction in infectivity of approximately 40% for RNA viruses, whereas only an 11% reduction was noted for the DNA virus. Remarkably, the infectivity of RNA viruses was totally eradicated after 12 h of contact with the ZnO nanolayer. In the case of anaerobic bacteria-laden bioaerosols, inhibition ratios ranged from 58% to 97% across various anaerobic strains, while aerobic bacteria aerosols demonstrated inhibition ranging from 26% to 74%. Notably, after 24 h of direct contact between bacteria and ZnO nanolayer, a substantial viability inhibition of most strains (80%–90%) was achieved. These findings underscore the potential of ZnO nanolayer for diverse biomedical purposes, encompassing personal protective equipment and other applications such as air purification systems.