Many lactic acid bacteria act as probiotics and are useful for preventing or maintaining the balance of intestinal microbiota. Probiotic bacteria have the ability to colonise the colon by adhesion to the intestinal mucosa and thereby prevent pathogens from attaching by competitive exclusion. Bacteria of the genus Lactobacillus are potential probiotic strains, especially those expressing S-proteins because they are in direct contact with the bacterial environment and can therefore be included in numerous surface properties. The role of S-protein in the competitive exclusion of the pathogenic bacteria Salmonella enterica serovar Typhimurium FP1 with strains Levilactobacillus brevis MB1 and MB2, isolated from maternal milk, has been studied in this work. Adhesion of the same probiotic strains to intestinal Caco-2 epithelial cells and to extracellular matrix proteins as well as the role of S-protein in unfavourable gastrointestinal tract conditions was further investigated. S-proteins present on the cell surface of the tested probiotic strains MB1 and MB2 have positively contributed to the competitive exclusion of the test microorganism Salmonella Typhimurium FP1 under in vitro conditions. The positive effect of S-protein was also observed during the adhesion of bacterial cells to extracellular matrix proteins since the number of bacterial cells after treatment with guanidine hydrochloride was decreased. Cell survival of bacterial strains MB1 and MB2 under unfavourable GIT conditions was higher in the presence of S-protein.