Physical adsorption has shown to be facile and highly effective to deposit chitosan nanowhiskers (CsNWs, 60 % deacetylated, length: 247 nm, thickness: 4-12 nm, width:15 nm) on electrospun cellulose acetate nanofibers (CANFs, 560 nm) to effect complete surface charge reversal from negatively charged CANFs (-40 mV) to positively charged CsNWs-adsorbed CANFs (+8 mV). The CsNWs coverage did not alter the smooth and homogeneous morphology of fibers, as observed from SEM images. Biological assays showed the CsNWs covered nanofibers were effective against the Gram-negative bacterium E. coli, reducing 99 % of colony forming units (CFU) in 24 h and atoxic to healthy Vero cells. The use of CsNWs to modify cellulose fiber surfaces has been proved to be efficient and may be applied to a broad scope of fields, especially as biomaterials and biomedical applications.