Chitosan non-woven fabrics were acetylated to improve their antimicrobial properties. The active chlorine content, antimicrobial properties, storage stability, and surface properties of acetylated chitosan non-woven fabrics were investigated. The active chlorine content of the fabrics increased upon reduction of the degree of the acetylation or increase in sodium hypochlorite concentration. Acetylated chitosan non-woven fabrics showed powerful antimicrobial activity by efficiently killing Escherichia coli and forming a growth inhibition zone for Staphylococcus aureus. Furthermore, scanning electron microscopy observations demonstrated that the acetylated chitosan non-woven fabrics were not damaged in sodium hypochlorite solution.