An alternative approach to application of chitosan based on layer by layer (LbL) assembled technique is studied in this paper. For this aim, chitosan (CHT) was used as a bio-based cationic polyelectrolyte and pentasodium tripolyphosphate (TPP) and poly(sodium 4-styrene sulfonate) (PSS) were selected as anionic polyelectrolyte. TPP/CHT and PSS/CHT based bilayers were fabricated on the cationized woven cotton fabrics via layer-by-layer self-assembly technique. The characterization of coatings on the fabric surface in terms of surface appearance, atomal content, and chemical bondings were made in detail through SEM, XPS, and FTIR-ATR analysis. Also, the antibacterial activity, air permeability, and water contact angle were measured. Surface analyses demonstrate the interaction between TPP, PSS and chitosan separately. XPS spectra also showed the existence of LbL deposition over cotton substrates in terms of both elemental composition and the presence of different types of bondings on the fabric surface. The antibacterial activity analysis revealed that the modified cotton fabric with the addition of CHT/TPP and CHT/PSS bilayers could increase the degree of inhibition on K. pneumanie and S. aureus bacteria.