Lactobacilli, the most commonly used probiotics, show strain specific immunostimulatory effects. The strain specificity is related to the structure of cell-wall components such as lipoteichoic acids (LTAs), ligands for TLR2. In contrast, the role of exopolysaccharides (EPSs), the major components of lactobacilli biofilm, in the “cross-talk” between bacteria and the immune system is poorly documented. In this study, we have tested immunomodulating potential of lactobacilli derived EPSs, by their ability to modulate in vitro production of cytokines by murine peritoneal macrophages. We have examined EPSs of three lactobacilli strains (L. reuteri, L. johnsonii, L. animalis/murinus) isolated from colitic mice. The effect was compared with that of whole bacteria and LTA. All three nonviable Lactobacillus strains induced high amounts of cytokines with a strain specific profile. In contrast, the tested exopolysaccharides, exert low, albeit highly strain diversified stimulatory activities. The immunostimulatory potential of EPSs was correlated with the interaction strength between EPS and macrophage receptors as measured by atomic force spectroscopy.