A novel nanocomposite film (Cs/FeS film) for potential food packaging applications was fabricated using chitosan (Cs) and Bio-FeS biosynthesized by Deinococcus wulumuqiensis R12. The impregnation of chitosan with Bio-FeS nanoparticles was evidenced by the EDS mapping, FTIR and XRD. Compared with pure chitosan films, the Cs/FeS film exhibited improved shading performance, better water resistance, as well as high antioxidant capacity, making it an attractive option for food packaging. With the increase of Bio-FeS content, the physical properties were further enhanced to a certain degree. Furthermore, Bio-FeS nanoparticles were found to simultaneously endow Cs/FeS films with increased elasticity and ductility at a low filler content of 0.4%. In addition, Cs/FeS films exhibited excellent antimicrobial activity against Salmonella typhimurium (97.7% inactivation efficiency) and Staphylococcus aureus (99% inactivation efficiency). At the same time, the peroxidase-like properties of Bio-FeS enable the chitosan film to respond to H2O2 in food. Notably, chitosan film impregnated with Bio-FeS greatly outperformed commercial plastic wrap in terms of its ability to preserve meat, indicating its potential use in the development of novel preservation methods.