In this paper, we describe the preparation and characterization of colloidal Cu nanoparticles/chitosan composite film (composite film) by solution-casting technique with microwave heating. Effects of the incorporation of colloidal Cu nanoparticles on structure, thermal behavior, surface, barrier properties and light transmission of composite film were investigated. The antimicrobial activity of films against Staphylococcus aureus and Salmonella enterica serovar Typhimurium, were also tested. Incorporation of colloidal Cu nanoparticles on chitosan matrix improved the barrier properties of films, decreasing the oxygen permeability as well as water vapor permeability and increasing the protection against UV light. The composite film was effective in alteration of cell wall and reduction of microbial concentration in the liquid culture for both bacteria tested.