Evolution of new materials that offer protection for active food ingredients is the focal point for food application research. Release of antimicrobial compounds from the packaging film is one of the common methods. In this research, an antimicrobial active packaging film based on poly(vinyl alcohol) (PVA) and thymol was prepared via freezing/thawing technique. The effect of thymol content on the property and structure of the film was characterized by Fourier transform infrared spectroscopy (FT-IR), Scanning electron microscopy (SEM), thermogravimetric analysis (TGA), X-ray diffraction (XRD), swelling and gel fraction measurements. Furthermore, the antimicrobial test shows that the prepared film has high antimicrobial effect against Gram-positive bacterial strains (Bacillus subtilis), fungal strain (Candida albicans), and Gram-negative bacterial strains (Salmonella senftenberg and Escherichia coli) in comparison with the control PVA film. The current approach verified that PVA film containing thymol could be applied as an alternative for food backing applications