Chitosan/ZnO (CS/ZnO) nanocomposite materials have recently attracted intensive research interest because of their combined ZnO bactericidal activity and good biological compatibility of CS. In this study, ZnO NPs were synthesized via a green process using orange peel extract as a reducing agent. The CS/ZnO nanocomposites were chemically synthesized with different CS/ZnO weight ratios. The microstructure, morphology, and thermal stability of the nanocomposite materials greatly depended on the weight ratio of CS/ZnO. The nanocomposite materials exhibited strong antimicrobial activity towards both Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus) in the absence of UV light at all of the investigated CS/ZnO ratios. Due to the different characteristics of bacteria types, the activity inhibition towards E. coli was nearly constant in all samples, while that towards S. aureus slightly decreased with the increment of chitosan content. The results of this study suggest that the nanocomposites have potential applications in controlling the spread of infection by various bacteria.