Due to the distinctive characteristics of probiotics, it is essential to pinpoint strains originating from diverse sources that prove efficacious in addressing a range of pathologies linked to dysfunction of the intestinal barrier. Nine strains of lactic acid bacteria were isolated from two different sources of tepache kefir grains (KAS2, KAS3, KAS4, KAS7, KAL4, KBS2, KBS3, KBL1 and KBL3), and were categorized to the genus Lacticaseibacillus, Liquorilactobacillus, and Lentilactobacillus by 16S rRNA gene. Kinetic behaviors of these strains were evaluated in MRS medium, and their probiotic potential was performed: resistance to low pH, tolerance to pepsin, pancreatin, bile salts, antibiotic resistance, hemolytic activity, and adhesion ability. KAS7 strain presented a higher growth rate (0.50 h-1) compared with KAS2 strain, who presented a lower growth rate (0.29 h-1). KBS2 strain was the only strain that survived the in vitro stomach simulation conditions (29.3%). Strain KBL1 demonstrated significantly higher viability (90.6%) in the in vitro intestine simulation conditions. Strain KAS2 demonstrated strong hydrophilic character with chloroform (85.6%) and xylol (57.6%) and a higher percentage of mucin adhesion (87.1%). However, strains KBS2 (84.8%) and KBL3 (89.5%) showed the highest autoaggregation values. In terms of adhesion to the intestinal epithelium in rats, strains KAS2, KAS3 and KAS4 showed values above 80%. The growth of the strains KAS2, KAS3, KAS4, KBS2, and KBL3 was inhibited by cefuroxime, cefotaxime, tetracycline, ampicillin, erythromycin, and cephalothin. Strains KBS2 (41.9% and 33.5%) and KBL3 (42.5% and 32.8%) had the highest co-aggregation values with S. aureus and E. coli. The results obtained in this study indicate that lactic acid bacteria isolated from tepache can be considered as candidates for potentially probiotic bacteria, laying the foundations to evaluate their probiotic functionality in vivo and thus to be used in the formulation of functional foods.