In this study, the characterization of some bioactive content of exopolysaccharide (EPS) extracted from Pediococcus ethanolidurans isolated with Smilax excelsa L. from conventionally produced pickles were investigated. Background: Although this study is the main study involving the characterization of EPS obtained from Pediococcus ethanolidurans, it is important because it has determined a natural polysaccharide that can be used in different fields in the industry. According to the obtained results, sugar analysis by GC-MS revealed that EPS consisted of glucose (7.59%), mannose (41.96%), fructose (16.98%), arabinose (3.15%) and rhamnose (30.30%). From the thermal behavior determined by differential scanning calorimetry (DSC), it was concluded that it should not be heated close to 250 °C. At the same time, according to the thermogram obtained as a result of X-ray diffraction (XRD) analysis, it was found to have a crystalline structure. EPS, which reached 73.844% efficiency, showed antibacterial activity against Listeria monocytogenes, Staphylococcus aureus and Escherichia coli, while 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) scavenging activity was detected at 0.24 mM. It also has remarkable results, seen in cytotoxicity analysis against healthy HT-29 cells, demonstrating that it has proliferative activity as high as %125 In short, Pediococcus ethanolidurans was found to be a novel EPS producer with impressive properties.