Nanoparticle metal oxides are emerging as a newclass of important materials inmedical, agricultural, and industrial applications. In this context, free zinc oxide (ZnO) nanoparticles (NPs) have been increasingly shown with broad antimicrobial activities.However, biological properties of immobilized ZnO NPs on matrixes like nanofibrous membranes are still limited. In this study, in situ synthesized ZnO NPs/polyvinyl alcohol (PVA) nanofibrous membranes were fabricated by electrospinning with different zinc acetate concentrations. Characterization results indicated that, with 5mM zinc acetate, uniform size ZnO NPs (~40 nm) were formed and evenly distributed on themembrane surface.The surfaces became more hydrophobic with higher concentration of zinc acetate. ZnO NPs/PVA nanofibrous membranes showed a broad spectrum of antimicrobial activities and cell adhesion inhibiting effects against four microorganisms including Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli, fungi Candida albicans, and spores of Aspergillus Niger. Our data revealed that the major antimicrobial mechanism could be attributed to cell membrane damage and cellular internalization of ZnO NPs, while the hydrophobic surface of themembrane primarily contributed to the cell adhesion inhibition. This study suggests that ZnO NPs/PVA nanofibrous membranes could potentially be used as an effective antimicrobial agent to maintain agricultural and food safety.