Post-operative adhesions are one of the most important problems faced by patients and surgeons. In this study, nanofibrous mats were produced as novel surgical adhesion barrier from polysaccharide-based polymers, hyaluronic acid, carboxymethyl cellulose and sodium alginate, via electrospinning. The produced nanofibrous mats were crosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide. Furthermore, the morphology, in vitro degradation, cytotoxicity and cell adherence potentials of the nanofibrous mats aimed to be used as adhesion barriers were evaluated and compared with a commercial adhesion barrier. Results of the in vitro experiment showed that the nanofibrous mats have maintained their physical structures during the critical period for adhesion formation, and had non-adherent cell feature and non-cytotoxic nature required for an ideal adhesion barrier.