The inappropriate use of antimicrobials has resulted in the selection of resistant strains. Thus, a great number of studies have focused on the investigation of new antimicrobial agents. The use of zinc oxide nanoparticles (ZnO NPs) to optimise the fight against microbial resistance has been receiving increased attention due to the non-specific activity of inorganic antimicrobial agents. The small particle size and the high surface area of ZnO NPs can enhance antimicrobial activity, causing an improvement in surface reactivity. In addition, surface modifiers covering ZnO NPs can play a role in mediating antimicrobial activity since the surface properties of nanomaterials alter their interactions with cells; this may interfere with the antimicrobial effect of ZnO NPs. The possibility of using surface modifiers with groups toxic to microorganisms can improve the antimicrobial activity of ZnO NPs. Understanding the exact toxicity mechanisms is crucial to elucidating the antimicrobial activity of ZnO NPs in bacteria and fungi. Therefore, this review aims to describe the mechanisms of ZnO NPs toxicity against fungi and bacteria and how the different structural and physical-chemical characteristics of ZnO NPs can interfere in their antimicrobial activity.