The aim of this research was to compare the effect of free and immobilized Lysozyme on the physicochemical and antimicrobial properties of sodium caseinate (SC) based active films. Lysozyme was immobilized onto bacterial cellulose nanofiber (BCNF) and decreasing of its activity was approved after immobilization. Free and immobilized enzymes were incorporated into SC films at the concentrations of 0.5 and 1 mg.100ml-1 and the films were characterized. Addition of BCNF and lysozyme diminished significantly (p˂0.05) the moisture absorption and water vapor permeability of SC films and immobilized enzyme had higher effect than free enzyme on decreasing of these parameters. The tensile strength and Young’s modulus were increased and elongation to break was decreased by incorporation of BCNF. An adverse effect was observed for lysozyme addition but the effect of immobilized enzyme on the weakening of tensile properties was lower than free lysozyme. According to XRD results, the crystallinity of films increased by incorporation of BCNF and lysozyme. However, the immobilization reduced the crystalline regions of BCNF. Antimicrobial activity of lysozyme increased after immobilization and SC films containing immobilized enzyme exhibited considerable activity against Gram-positive bacteria S.aureus, L.monocytogenes. Gram-negative bacteria E.coli, Y.enterocolitica, mold of A.niger and yeast of S.cerevisiae.