Gelatin films were prepared by enzymatic cross-linking with transglutaminase, and their mechanical and barrier properties were evaluated as functions of relative humidity (RH, 30 to 75%), temperature (15 to 35°C) and glycerol content (15 to 31%). Water and glycerol plasticized the films synergistically, resulting in greater elongation but lower tensile strength values. Films with higher glycerol contents exhibited higher moisture contents, indicating higher hydrophilicity of the films. Permeabilities of oxygen and allyl isothiocyanate (an antimicrobial vapor from Cruciferae plants) were low when the films were dry, but increased considerably when RH>50%. Therefore, RH conditions during end-use applications must be considered to optimize the performance of these films.