The objective of this work was to better display the effect of electron beam accelerator doses (0, 20, 40 and 60 kGy) on structural, mechanical and barrier properties of edible lightly plasticized chitosan-fish gelatin blend film. From Electron Spin Resonance (ESR), signal at 3500 G for blend film was identified as free radical formation during irradiation, which might introduce intermolecular cross-linking into the polymer matrix, thus affecting structural properties. Tensile Strength (TS) for gelatin film significantly increased with growing irradiation doses (improved by 30% for 60 kGy), but the TS of chitosan and blend films were not significantly affected. On the contrary, irradiation significantly reduced elongation at break (%E) for chitosan and blend film up to 50% whereas it twice increased the Young modulus. Moisture barrier efficiency (30–84% RH gradient) of blend films was slightly improved after irradiation. Oxygen permeability also decreased after 60 kGy irradiation treatment, for both chitosan and blend films. Surface hydrophobicity tended to decrease after irradiation. From Fourier Transform Infra-Red (FTIR) spectra, some noticeable differences were observed after irradiation, in the relative intensity and position of bands in the region between 3600 and 2800 cm−1 and between 1700 and 1500 cm−1. UV-vis analysis shows that all films displayed an absorbance peak between 280 and 385 nm. These peaks are shifted toward higher wavelengths after irradiation This clearly showed some modifications in the interactions (hydrogen bonds, amide groups) between polymer chains induced by the irradiation.