One of the most commonly observed surface structures on the prokaryotic cell envelopes is monomolecular crystalline array of proteinaceous subunits termed Surface Layers or S-layers. Due their self assembly ability and the highly ordered, regular structure down to the nanometer scale, S-layers were demonstrated to possess a great potential for nanobiotechnological applications. Some probiotic bacteria, such as Lactobacillus spp. have been found to possess S-layers. In this perspective, the objectives of this study were isolation and identification of Lactobacillus species from traditional dairy products carrying surface (S) layer protein and investigation of important prerequisite of probiotic interest, such as the capability to survive at low pH and in presence of bile salts. The protein profile of intact Lactobacillus isolates was analyzed in SDS-PAGE. The protein bands of 45–60 kDa were present in two of the 25 isolates, suggesting the presence of S-layer proteins in these strains. The above mentioned proteins were recovered in the guanidine hydrochloride extracts. After dialysis, the extracted proteins showed a significant band of apparent molecular mass of 50–60 kDa in SDS-PAGE. The strains possessing S-layers protein was identified both biochemically and by 16S rRNA sequencing. The 16S rRNA gene sequence analysis revealed that two isolates exhibited maximum similarity with the 16S rRNA sequence of Lactobacillus brevis spp. These two isolates were resistant to acid and bile in which their viability in acidic condition were between 51.6% and 77.8% and bile resistance of two isolates was approximately 99% after 24 h.