Exopolysaccharides are postbiotics secreted by lactic acid bacteria that contribute to bountiful health-promoting properties. In the present investigation, exopolysaccharide (EPS 4412) extracted and purified from Pediococcus pentosaceus 4412 was subjected to structural characterization such as scanning electron microscopy, atomic force microscopy, x-ray diffraction, and thermogravimetric analysis which depicted the EPS 4412 as a porous fibrous microstructure, irregular and smooth surface, semi-crystalline, with thermal stability at 252.86°C. Anti-biofilm activity of EPS 4412 exhibited the maximum adhesion inhibition against Staphylococcus aureus (76.07 ± 2.03%), Bacillus cereus (71.86 ± 0.89%), Salmonella enterica (50.5 ± 0.57%), and Shigella flexneri (38.84 ± 0.38%) at 4mg/mL concentration. At 10 mg/mL concentration, the EPS 4412 displayed moderate DPPH radical scavenging activity of 55.62 ± 0.85% and a reducing power of 1.54 ± 0.03 at OD700 nm. In vivo, the anti-inflammatory activity of EPS 4412 in LPS-induced C57BL/6 mice revealed elevated body weights, thymus, and spleen indices, increased glutathione peroxidase and catalase activities, and reduced malondialdehyde levels. The EPS 4412 had substantially upregulated anti-inflammatory IL-10 and downregulated the pro-inflammatory cytokines, IL-6, and TNF-α to alleviate the inflammatory process. Histopathological examination of the intestine, liver, and spleen tissues disclosed the protective effect of EPS 4412. Thus, EPS from Ped. pentosaceus 4412 has the potential to regulate inflammatory responses, alleviate oxidative stress, and protect organs. EPS 4412 could be employed as a natural food additive and anti-inflammatory drug in the food and pharmaceutical industries.