Nowadays, polymers obtained from edible resources such as polysaccharides and proteins have attained growing consideration to substitute petroleum-derived polymers. Edible films are a good alternative for the plastic packaging since these films are biodegradable, environmental-friendly and non-toxic. However, protein based edible films have poor water permeability and lower mechanical strength. In this study, edible films based on whey protein isolate (WPI) were produced with different microbial transglutaminase (MTG) concentrations (0, 5, 10 and 15 Unit/g of protein) and the effect of enzymatic treatment on the film properties was investigated. Results showed that as compared to control, the treatment with the lower concentration of MTG (5–10 U/g) significantly increased the tensile strength (TS) and decreased elongation at rapture (ER) values of WPI-films, while at the higher concentration of MTG (15 U/g), TS value slightly decreased and ER values significantly increased. By increasing the enzyme concentration from 5 to 10 U/g, water vapor transferability (WVT) and water soluble fractions (WSF) decreased significantly (P≤0.05). The MTG-treated films except the film treated with 15 U/g TG, had homogeneous and even surface without any crack or fracture. Based on results, treatment with MTG enzyme may be applied as an appropriate technique to modify the structural and barrier properties of WPI-based films.