This study was performed to check the effect of different essential oils on chitosan and gelatin-based antimicrobial films. Films prepared from biopolymers contain better mechanical strength but lack in moisture barrier properties. In order to increase the moisture barrier properties of chitosan and gelatin-based films in the current research work, different essential oils, i.e., thyme, cinnamon, basil, ginger, and cumin, at varying concentrations (1.0, 1.5, and 2.0%) were incorporated. Moreover, the concentrations of glycerol (plasticizer) and emulsifier (Tween 20) were kept constant to maintain homogeneity in the research. Antimicrobial films composed of gelatin and chitosan infused with essential oils were evaluated for their physicochemical (emulsion stability, particle size, and viscosity), FT-IR, microstructural (scanning electron microscopy), moisture barrier (water vapor permeability), and antimicrobial properties (E. coli, Salmonella, and S. aureus). Study outcomes elucidated significant variations (p < 0.05) as the concentration of essential oil was increased in the film solutions. An increased concentration of essential oil (2.0%) significantly enhanced the moisture barrier properties (1.12 ± 0.03 g.mm/kPa.h.m2). Nevertheless, the tensile strength decreased (38.60 ± 1.4 to 31.50 ± 1.5 MPa) from 1 to 2%. The increase in essential oil concentration in the emulsion-based films also influenced their physicochemical characteristics, such as droplet size, viscosity, and emulsion stability. At lower concentrations (1.0%), films exhibited a uniform microstructure but lacked moisture barrier properties. Antimicrobial properties against E. coli, Salmonella, and S. aureus showed an increased inhibition effect as the concentration of essential oil was increased. Of the essential oil-based films, ginger- and basil-based films showed greater inhibition effects as compared to the other essential oils. Overall, antimicrobial films containing a 1.5% concentration of ginger and basil oil showed better results as compared to the other treatments for mechanical, moisture barrier, and antimicrobial properties, while films with a 2.0% oil concentration showed better antimicrobial and moisture barrier properties but lacked in mechanical properties. Essential oil-based antimicrobial films have prospective applications in foods, specifically in fresh and processed food items such as seafood, meat, chicken, and sausages.