Exopolysaccharides (EPS) produced by lactic acid bacteria are complicated polymers with industrial applications. LAB were isolated, screened for EPS production, and their probiotic properties determined. The anti-biofilm activity of EPS was investigated. Safety of EPS-producing isolate was investigated and it was molecularly identified through 16S rRNA sequencing. Finally, anti-biofilm and emulsification activity of EPS was studied and it was characterized using FT-IR, TGA, ¹H-NMR, DLS and HPLC. Thirteen LAB were isolated from dairy products. They showed probiotic characteristics like acid resistance (0–6.51 CFU ml⁻¹) hydrophobicity (8–54.04%), autoaggregation (0% [t = 2 h]–99.8% [t = 24 h]) and coaggregation with food borne pathogens. Among them, Enterococcus durans DU1 had ability to produce EPS. EPS of Enterococcus durans DU1 showed antibiofilm activity against Y. enterocolitica (24.06–51.36%), S. aureus (12.33–49.6%), and B. cereus (11.66–27.16%). FT-IR showed this EPS had characteristic absorption peaks due to the presence of the pyran ring of sugars. ¹H NMR showed that EPS has N-acetyl, methyl, and alkyl groups in its structure. The HPLC analysis showed that EPS is a heteropolysaccharide and consists of sucrose, glucose, and fructose. EPS showed significant thermal stability (20% weight loss) under 300 °C and zeta potential of − 18.1 mV. This EPS can be used in the food industry with no adverse effect on consumers