The aim of this work is to investigate the colonization potential and effect on the composition of the intestinal microbiota of S-layer carrying Lactobacillus brevis SF9B and plantaricin producing Lactobacillus plantarum SF9C in vivo on the model of experimental rats. Two different approaches were applied for the analysis of the intestinal microbiota composition. PCR-DGGE method was employed for assessing the presence of selected Lactobacillus strains, after their transit through the gastrointestinal tract (GIT) of the rat, while next-generation sequencing via Illumina MiSeq platform was applied for determination intestinal microbiota composition and effect of the selected strains in the, by amplifying V3 and V4 16S rRNA gene regions. PCR-DGGE analysis revealed the presence of both examined strains as well as detection of other Lactobacillus species representatives, the identified as Lactobacillus animalis, Lactobacillus intestinalis and Lactobacillus reuteri, which are autochthonous bacteria in the rat intestine, even 3rd and 10th day after the end of application. The metagenomic analysis confirmed an abundant prevalence of Lactobacillus spp. was in the gut microbiota of rats inoculated with SF9C and SF9B strains compared with the control group, even 10 days after the end of treatment. The results of this research supported enhanced functionality potential of jointed application of SF9C and SF9B strains, where both strains can benefit, Lb. plantarum SF9C by facilitated adhesion due to the competitive pathogen exclusion by coexisting Lb. brevis SF9B, while in same SF9B benefits in improved colonization due to the plantaricin production by Lb. plantarum SF9C, resulting altogether in a broader spectrum of antimicrobial activity against pathogens of the coculture.