Despite numerous investigations during recent decades in the field of antimicrobially treating textile fibers using chitosan, many obscurities remain regarding the adsorption/desorption behavior of chitosan and the influences of the accessible amino group content on the antimicrobial effectiveness of chitosan-treated fibers. Accordingly, the principal aim of the presented research was to evaluate different regenerated cellulose fibers regarding their abilities to provide highest antimicrobial effectiveness after chitosan treatments. Regenerated cellulose fibers such as viscose, modal and lyocell were antimicrobial functionalized by chitosan from solutions. The adsorption and desorption of the chitosan were analyzed using potentiometric titration, the spectrophotometric method with C.I. Acid orange VII, the Kjeldahl technique and polyelectrolyte titration. Mechanical properties of functionalized fibers were also checked. The antimicrobial activities of chitosan-functionalized cellulose fibers were examined, with regard to pathogen bacteria and fungi. The highest amino group content as a consequence of more intensive chitosan adsorption was found in viscose followed by modal and, finally, by lyocell. It was confirmed that those fibers with higher content of amino groups demonstrated a better reduction in pathogenic bacteria as well as pathogenic fungi. The functionalized cellulose fibers displayed potential utility in different fields of application, as exemplified by medical textiles for single use.