Chitosan is applied for novel and multifunctional biomaterials development. To enhance physicochemical and biological properties, as well as to improve the functionality of chitosan-based materials, multiple natural products are incorporated, such as essential oils. Essential oils with therapeutic potential can also be extracted from natural resins. Mastic gum, a soft resin collected mainly from Pistacia lentiscus, is a source of mastic oil, which is characterized by antimicrobial, antioxidant, anticancer activities, and wound healing properties. The purpose of the study was to prepare and physicochemically characterize chitosan films enriched with mastic oil and polysorbate 80 as an emulsifier. FTIR spectroscopy was used to evaluate chemical structure. Mechanical properties, swelling degree, and contact angle were also investigated. The effect of mastic oil and emulsifier on the properties of chitosan films was observed. Modified films incorporated with various concentrations of mastic oil were characterized by the hydrophilic surface, increased tensile strength, and elongation at break, thus greater flexibility as well as swelling capacity and stability in phosphate-buffered saline. These features might be an advantage in terms of material fabrication intended for biomedical applications, for example, wound healing. Chitosan and mastic oil might be a promising combination for use in biomaterials.