In this research, two N-halamine polymer precursors, a cationic homopolymer poly((3-acrylamidopropyl)trimethylammonium chloride) (CHP) and an anionic homopolymer poly(2-acrylamido-2-methylpropane sulfonic acid sodium salt) (AHP), have been successfully synthesized and coated onto cotton fabrics via a layer-by-layer (LbL) deposition technique. The coated cotton fabrics were characterized using scanning electron microscopy, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy. The biocidal efficacies of uncoated and coated cotton fabrics were evaluated against Staphylococcus aureus and Escherichia coli. The chlorinated swatches (CHP-Cl and AHP-Cl) inactivated 100% S. aureus and 99.73% E. coli O157:H7 in 30 min. Over 51% of the chlorine is retained after the equivalent of 50 machine washes. A skin stimulation test showed that CHP-Cl and AHP-Cl compounds have no irritation to rabbit skin, and so these swatches might be utilized for biomedical applications in the future. As an easy and efficient way of coating fabrics, the LbL deposition technique can broaden the use of N-halamine biocides in other polar substances as antimicrobial functional coatings.