Layer-by-layer self-assembly technology was introduced with adsorption of oppositely charged polycations and polyanions. The composite thin film consisting of quaternized chitosan derivative/poly(2-acrylamide-2-methylpropane sulfonic acid sodium salt) was created on the surface of cotton fabrics by using this method without much damage for breaking strength of swatches. The oxidative chlorine of five bilayers deposited on the swatches could reach 0.25%. The treated cotton fabrics exposure to dilute household bleach possessed excellent antimicrobial property, which could inactivate 100% of Staphylococcus aureus and Escherichia coli O157:H7 bacteria with a contact time of 1 min. The chemical surface modification, surface morphology, washing stability, storage stability, and mechanical property of the modified swatches were characterized and confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy, atomic force microscopy, stability measurement, and tensile strength test, respectively.