Cellulose acetate (CA) films with sweet fennel essential oil (FEO) were evaluated for possible changes caused by the incorporation of 5, 10, 20 and 30% glycerol. The results show that the incorporation of different concentrations of plasticizer caused an increase in thickness, water vapor transmission rate (WVTR), tensile strength (TS), besides altering the optical properties and demonstrating possible chemical interaction with the CA matrix (Fourier transform infrared (FTIR)). Scanning electron microscopy (SEM) images revealed that the addition of glycerol caused morphological changes on the surface and internal region of all films. As for antimicrobial activity, the FEO was effective for Staphylococcus aureus and Escherichia coli. However, all films evaluated did not show activity in inhibiting these microorganisms. Therefore, it is believed that the FEO may have some incompatibility with the CA matrix, being trapped between the polymer chains. Therefore, the results suggest that the incorporation of glycerol caused changes in the functional properties of all films, although it did not result in measurable antimicrobial effects.