Chitosan is a promising candidate as an antimicrobial agent in food packaging materials and biomaterials due to its biocompatibility, biodegradability, non-toxicity and biocidal activity. In this study, chitosan derivatives, α-(5,5-dimethyl hydantoin amino)-chitosan (CS-HDH), were synthesized. The CS-HDH nanospheres were prepared by ionic gelation method and characterized by SEM, FT-IR, XRD, and TGA-DTG. The prepared novel chitosan nanospheres in the range of 200–300 nm had good dispersibility. The CS-HDH nanospheres were used to prepare antibacterial PVA hybrid films by solvent evaporation technique. The surface morphology and thermal property of hybrid films were measured by SEM, AFM, and TGA-DTG. The results of antibacterial test showed that the hybrid film with 1.24 × 1018 atoms/cm2 of active chlorine exhibited excellent antibacterial properties against Staphylococcus aureus and Escherichia coli O157:H7, and all of bacteria could be inactivated within 5 min.