The objective of this research was to explore the functional qualities of two EPSs produced by recently discovered LAB (Lactobacillus delbrueckii (EPS-LB3) and Lacticaseibacillus rhamnosus (EPS-MLB3)) that have potential probiotic benefits. The study involved evaluating their biological characteristics, such as their antioxidant, antidiabetic, antimicrobial, antibiofilm, and antiproliferative activities at various concentrations, as well as investigating their effects on the gut microbiome through in vitro fecal fermentation. Moreover, the study analyzed the rheological properties of the EPSs in fermented bovine milk. The average molecular weights of the extracted EPS were 3762.43 kDa and 1272.19 kDa with monosaccharide compositions of Glu:Rib:Man:Xyl (1.0:16.4:6.6:6.5) and Rib:Man:Xyl:GA:Ara (7.1:1.6:4.8:1.0:9.0) for EPS-LB3 and EPS-MLB3, respectively. EPS-LB3 and EPS-MLB3 at 250 mg/L showed scavenging rates of 34.0 ± 1.7 % and 37.5 ± 1.1 % for DPPH, 47.3 ± 0.8 % and 56.6 ± 0.7 % for ABTS, 38.3 ± 0.5 % and 43.5 ± 0.6 % for SD, 53.9 ± 0.1 % and 54.7 ± 0.1 % for SAS, 10.6 ± 0.1 % and 10.7 ± 0.2 % for HP, 88.8 ± 0.1 % and 84.8 ± 0.5 % for HRS, 80.0 ± 1.4 % and 84.5 ± 0.8 % for MC, as well as 60.6 ± 1.7 % and 58.1 ± 0.9 % for Lipid Oxidation, respectively, suggesting good antioxidant properties. They also exhibited antimicrobial and anti-biofilm effects against several foodborne pathogens, and antiproliferative activities against cancer cell lines. Additionally, the utilization of EPS by several probiotics indicated the prebiotic nature of EPS. The effect of both EPS on gut microbiome by fecal fermentation revealed that these EPS promoted selective bacteria like Faecalibacterium prausnitzii and Ruminococcus bromii in the gut, which are responsible for carbohydrate metabolism and short-chain fatty acid production.