This work aimed to investigate the effect of EPS (extracellular polysaccharide) of Weissella cibaria as a prebiotic to promote the growth and antibacterial properties of Lactobacillus rhamnosus. The morphological, growth behavior, and antibacterial properties of L. rhamnosus were determined in MRSB (de Man Rogosa Sharpe broth) supplemented with different concentrations of EPS (0.1-2%). The results revealed that the incorporation of the EPS (2%) in MRSA improved the bacterial growth in terms of colony-forming unit (CFU, 0.7 × 105 CFU/mL) compared to L. rhamnosus grown in bare MRSA. The SEM observation revealed that EPS incorporation in the MRSB culture media does not affect the morphological properties of L. rhamnosus. Moreover, it was confirmed that the extract of probiotics cultured in MRSA supplemented with EPS (2%) was exhibited strong antibacterial and antibiofilm activity against targeted pathogens. This L. rhamnosus extract was found to be biocompatible evidanced by erythrocyte hemolysis assay. These results confirmed that EPS regulates the growth of probiotics, resists pathogen infection, and biocompatibility. © 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.