Positively charged N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) and negatively charged soy protein isolate (SPI) were alternately assembled on cellulose acetate (CA) electrospun nanofibrous mats via electrostatic layer-by-layer self-assembly technique. CA nanofibrous mats coated with bilayers of HTCC and SPI possessed more orderly-arranged structure than uncoated CA mats according to the observation from scanning electron microscopy images. The average diameter of the nanofibers was enlarged by the increase of the bilayer number. X-ray photoelectron spectroscopy indicated that HTCC and SPI were coated on the surface of the CA mats successfully. The average diameters of inhibition zones of (HTCC/SPI)10.5-films-coated nanofibrous mats against Escherichia coli and Staphylococcus aureus were 9.6mm and 11.53mm, respectively, which demonstrated the highest antimicrobial activity among samples in presented study. Copyright © 2015 Elsevier Ltd. All rights reserved.