Poly (vinyl alcohol) (PVA) and chitosan (CH)-based blend films were prepared via a simple mixing and casting method. The films were characterized by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and ultraviolet-visible spectroscopy (UV-vis). Mechanical properties, thermogravimetric (TG), derivative thermogravimetric (DTG), and the inhibition of biofilm formation against Pseudomonas aeruginosa PAO1 (P. aeruginosa PAO1) of CH/PVA films were investigated. The results indicated that CH and PVA in the blend films interacted by physical crosslinks consisting of intermolecular hydrogen bonds and intermolecular hydrogen bonds with compatibility, and were more thermally-stable than that of PVA. CH addition decreased the tensile strength and the light transmittance. CH60:PVA40 film showed notable activity against adhesion and inhibited biofilm formation of P. aeruginosa PAO1, indicating that CH60:PVA40 film could be applied as an antimicrobial and biofilm inhibiting food packaging material.