Textiles with antimicrobial activity are necessary to prevent the action of pathogenic microorganisms on textiles, as well as their spread and transmission to protect the health of medical personnel and patients. There is a need for an application method that will provide textiles with the greatest resistance to the action of pathogenic microorganisms. This research presents data regarding the effect of chitosan application methods on bacteria reduction, which is useful for obtaining cotton yarns with antibacterial activity. Low molecular weight chitosan was used to pre-treated (alkaline scoured and bleached) cotton yarns in four application methods. Each application method changed the exhaustion, padding, drying, and rinsing phases to obtain antibacterial yarns against Staphylococcus aureus and Escherichia coli. Determining the applied chitosan amount and accessible amino groups was used to analyze their effect on antibacterial activity. Differences in surface morphology and chemistry between samples were analyzed by FTIR-ATR and SEM analysis. The result indicated that the methods in which the drying phase precedes the rinsing phase have a higher efficiency in reducing bacteria than the methods in which the rinsing phase precedes the drying phase. The method with the highest efficiency in reducing bacteria is the method in which the exhaustion phase is followed by a phases pad, dry, and rinse.