Gelatin is a kind of natural polymers that may be easily obtained by a controlled hydrolysis of the fibrous insoluble collagen in skins, bones and connective tissues of animals. It has excellent biodegradability, biocompatibility and good film forming properties. In this study, Zno nano-particles that are biocompatible and anti-bacterial were used. Gelatin/Zno nano composite films were prepared with different percentage of Zno nano-particles and exposed to glutaraldehyde vapor at different times. Thickness of the films was measured by the thickness gauge. Solubility and swelling test was carried out in order to determine optimum time of cross linking. According to the results, the optimum duration of cross-linking was 3 hours. UV visible spectroscopy, EDX elemental analysis and SEM mapping images were performed to investigate the release of Zno nano-particles, proving the presence and distribution of Zno nano-particles, respectively. The antibacterial test was carried out according to AATCC-147 standard test method to investigate the antibacterial properties of films. Results showed that nano composite film containing 0.5% of Zno nano particles had the most resistance against Staphylococcus aurous and Escherichia coli bacteria. The results of UV visible spectroscopy indicated the Zinc oxide nano-particles were released until 5 days of treatment.