The synthesis and functional characterization of an antibiofilm exopolysaccharide (EPS) from a probiotic Enterococcus faecium MC13 were investigated. The temperature of 35 A degrees C, pH of 6.5, and salinity of 1-2 % were found to be optimum for EPS production. The sucrose (30 g l(-1)) and yeast extract (20 g l(-1)) acted as suitable carbon and nitrogen sources, respectively, which strongly influenced EPS production with yield of 11.33 and 11.91 g l(-1). Based on the thin layer chromatography, EPS of E. faecium MC13 was found to be a heteropolysaccharide, composed of galactose and glucose sugar units with a molecular mass of 2.0 x 10(5) Da. Fourier transform infrared spectrum analysis of the EPS revealed many predominant functional groups including hydroxyl, carboxyl, and amide groups. EPS exhibited better emulsifying and flocculating activities which is relatively similar to those of commercial polysaccharides. In vitro antioxidant inspect of EPS showed lesser antioxidant activity than that of the control ascorbic acid. Thermal behavior of EPS was different from the other EPS produced by other lactic acid bacteria. In vitro antibiofilm assay of EPS exhibited significant biofilm inhibition, especially with Listeria monocytogenes. To the best of our knowledge, this is the first report on EPS of E. faecium with strong emulsifying and flocculating activities.