This work aimed to evaluate the antimicrobial effectiveness of a biodegradable composite, cellulose acetate film with the incorporation of various combinations of cinnamon, oregano and sweet fennel essential oils (EOs) against Escherichia coli, Staphylococcus aureus and Penicillium spp. Moreover, thickness, the mechanical and water vapor barrier properties of these films were evaluated. The films with the incorporation of pure oregano EO and the combination oregano + cinnamon EOs presented the best results against all microorganisms tested. The water vapor transmission rate of the film was reduced with the addition of the EOs and the thickness of the film increased. The EO increased the flexibility of the films, and the film with pure oregano EO was the most resistant, showed 30,9 N of puncture strength. The results suggest that the EOs incorporated as natural antimicrobial agents can potentially be used in food packaging, reducing the risk of microbial contamination of food.