Exopolysaccharides (EPSs) are novel functional additives for low-fat yogurt. Pharmaceutical, medical, and food industries are using more LAB-based EPSs. In this study, Leuconostoc spp. was used to produce ninth bacterial EPSs in a modified molasses medium. Production of EPSs was concentration-dependent on all stains and the highest yield was obtained from the S3 strain (55.23 g/l), followed by S6 (49.95 g/l), S8 (45.68 g/l), and S7 (44.23), respectively. HPLC and FTIR analysis showed that all purified EPSs from Leuconostoc citreum (S3) and Leuconstoc holzaapfelii (S8) were related to exopolysaccharide glucan. Anticancer activity of all EPSs samples (EPSs1-9) against Caco-2 cells and normal MCR-5 cells were investigated using MTT assay. The results revealed that Caco-2 cells were more sensitive than the normal MCR-5 cells. The highest anticancer activity against Caco-2 cancer cells was recorded for EPS8 (IC50 = 22.94 µg/ml, SI=3.73), followed by EPS3 (IC50 = 36.15 µg/ml, SI=8.72), EPS1 (IC50 = 50.01 µg/ml, SI=3.73), and EPS4 (IC50 = 94.90 µg/ml, SI=3.26), respectively. The lowest cytotoxicity was recorded for EPS5 (IC50 = 130.5 µg/ml). The most active EPSs (EPS3 and EPS8) were used as fat replacements and stabilizers in low-fat set yogurt at non-toxic concentrations (0.4, 0.8, and 1.2%). EPS3 and EPS8 improved the low-fat yogurt’s organoleptic and rheological properties. EPS8 had the highest water holding capacity (77.26%), viscosity (3660 CP), and lowest syneresis (22.95%) and whey off (0.6 ml). Low-fat set yogurt enhanced with EPS3 and EPS8 recorded the highest sensory evaluation values with overall acceptability, especially EPS3b, EPS3c, EPS8c, and EPS8b; the total score point of 97.50, 97.43, 96.51, and 96.36, respectively in fresh age compared to control yogurt (92.64). In conclusion, Leuconostoc EPSs, especially EPS8, can be explored for anti-cancer effects on Caco-2 colorectal cancer cells. It could also improve the rheological and organoleptic qualities of low-fat set yogurt.