This study aimed to assess the transit tolerance of potential probiotic dairy Lactobacillus strains in human uppergastrointestinal tract in vitro, and to evaluate the effect of EPS production on the viability and adhesion of these strains. Survival and adhesion of two exopolysaccharide (EPS)-producing L. delbrueckii subsp. bulgaricus strains (B3 and B2) and E. coli ATCC11229 were assessed after the exposure of different pH (gastric juice) and gastric plus pancreatic juice challenges. In the artificial gastric juice (pH 2), both the viability of the strain B3 and B2 was decreased. Artificial juice treatments significantly reduced the adhesion to caco-2 cells (P < 0.05). High EPS-producing B3 survived better in the adverse gastrointestinal conditions and showed better ability of adhesion to Caco-2 cells when assessed for competition with E. coli ATCC 11229 compared to low EPS-producing B2. This investigation showed that EPS production could be affected or be involved in the viability, adherence and competition of L. delbrueckii subsp. bulgaricus strains and support the potential of B3 strain for development of new probiotic products.