Sterilization through γ-irradiation has been reported to affect collagen mechanical properties, but its possible effects on gelatin based materials have not been investigated up to now. Herein we report the results of a mechanical, chemical and thermal study performed on gelatin films before and after γ-irradiation. The investigation was performed on uncrosslinked films as well as on crosslinked films. To this aim, two common crosslinking agents, glutaraldehyde and genipin, at different concentration (0.15, 0.30 and 0.67%) were used. The results indicate that sterilization significantly affects the mechanical properties of uncrosslinked films, whereas it displays a modest effect on gelatin swelling, release in solution, thermal stability and molecular structure. Both glutaraldehyde and genipin enhance the mechanical properties and stability in solution of the gelatin films. In particular, the values of Young modulus increase as a function of crosslinker concentration up to about 10 and 18 MPa for genipin and glutaraldehyde treated samples respectively. The results of in vitro study demonstrate that the films crosslinked with genipin do not display any cytotoxic reaction, whereas glutaraldehyde crosslinking provokes an acute and dose dependent cytotoxic effect.