Chitosan microspheres (CMS) by the emulsion-chemical cross-linking method with and without lysozyme immobilization were synthesized and characterized. The technique conditions were adjusted, and spherical particles with approximate diameters of 3.74 ± 1.08 μm and 0. 29 ± 0.029 μm to CMS and chitosan-lysozyme microspheres (C-LMS), respectively, were obtained. The microspheres were characterized by scanning electron microscopy (FESEM), Spectroscopy Fourier Transform Spectroscopy (ATR-FTIR), X-ray diffraction (XRD), and zeta potential. Particle size was identified by laser light scattering (DLS) and the thermal properties by Differential Scanning Calorimetry (DSC) and Thermogravimetry (TGA) were determined. By the lysis of Micrococcus lysodeikticus, the activity of the microspheres was determined, and the results correlated with the amount of lysozyme used in the immobilization process and the enzyme loading efficiency was 67%. Finally, release tests pointed out the amount of enzyme immobilized on the microsphere surface. These results showed that chitosan microspheres could be used as material for lysozyme immobilization by cross-linking technique. The antimicrobial activity was tested by inhibition percent determination, and it evidenced both chitosan microspheres (CMS) and chitosan-lysozyme microspheres (C-LMS) positive antimicrobial activity to Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa.