Chitosan microspheres are used in the biotechnological, biomedical, and enzyme immobilization fields. This study synthesized lysozyme-chitosan microspheres by crosslinking and covalent attachment. The synthesis conditions were adjusted, and spherical particles content lysozyme were obtained and characterized by Scanning electron microscopy (SEM) and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Activity and stability assays were conducted to determine the immobilization method's influence, and the kinetics parameters were calculated. The enzymatic activity recovery yields of 65.8% and 20.0% were obtained for crosslinking and covalent attachment, respectively. Surface lysozyme-chitosan microspheres' enzymatic activity was obtained by covalent attachment. By the crosslinking method, surface and into particle enzyme activity were observed. The functionalization of cotton fabric was achieved, and the impregnation slightly modified cotton fabric properties such as resistance and permeability. Finding antimicrobial properties against bacteria S. aureus and P. aeruginosa, with a growth of 49.12% and 65.31%, respectively, were obtained.