Many strains belonging to lactobacilli exert a variety of beneficial health effects in humans and some of the bacteria are regarded as probiotic microorganisms. Adherence and capabilities of colonization by Lactobacillus strains of the intestinal tract is a prerequisite for probiotic strains to exhibit desired functional properties. The analysis conducted here aimed at screening strains of Lactobacillus helveticus possessing a health-promoting potential. The molecular analysis performed, revealed the presence of a slpA gene encoding the surface S-layer protein SlpA (contributing to the immunostimulatory activity of L. helveticus M 92 probiotic strain) in all B734, DSM, T80, and T105 strains. The product of gene amplification was also identified in a Bifidobacterium animalis ssp. lactis BB12 probiotic strain. SDS-PAGE of a surface protein extract demonstrated the presence of a protein with a mass of about 50 kDa in all strains, which refers to the mass of the S-layer proteins. These results are confirmed by observations carried with transmission electron microscopy, where a clearly visible S-layer was registered in all the strains analyzed. The in vitro study results obtained indicate that the strongest adhesion capacity to epithelial cells (HT-29) was demonstrated by L. helveticus B734, while coaggregation with pathogens was highly diverse among the tested strains. The percentage degree of coaggregation was increasing with the incubation time. After 5 h of incubation, the strongest ability to coaggregate with Escherichia coli was expressed by T104. The T80 strain demonstrated a significant ability to co-aggregate with Staphylococcus aureus, while DSM with Bacillus subtilis. For B734, the highest values of co-aggregation coefficient was noted in samples with Salmonella. The capability of autoaggregation, antibiotic susceptibility, resistance to increasing salt concentrations, and strain survival in simulated small intestinal juice were also analyzed.