Biopolymer packaging materials such as zein have a high potential for replacement with synthetic polymers due to their biodegradability. Despite their environmental benefits, their application is not possible due to their poor properties. To overcome these limitations, using crosslinking materials such as essential oils (EOs) is a very effective method. Therefore, in this study, the effect of using Zataria multiflora EO (ZEO) on improving the properties of biodegradable zein film was investigated. Adding ZEO to the film had no effect on the film thickness. But, it reduced the tensile strength, water vapor permeability and brightness and increased the elongation at break, water solubility and a and b color parameters in film and improved the film surface properties according to AFM and SEM images. Also, the results of FTIR test indicated that new connections were established in the film structure due to the presence of EOs. Therefore, it seems that the use of zein biodegradable film containing ZEO is able to open a new window to the use of biodegradable materials in the food packaging industry in order to better maintain the quality and Open food safety and reduce the environmental hazards resulting from the accumulation of petroleum packaging materials.