Tepache is a fermented beverage of pre-hispanic origin and their consumption offers health benefits due to its probiotic content, including lactic acid bacteria and unconventional probiotics from the Bacillus genus. Three presumptive strains of Bacillus spp were isolated from Tepache, microbiological identification was conducted using selective culture media, initial taxonomic identification was performed using MALDI-TOF-MS with 16SrRNA gene sequencing and comparison against the GenBank database via BLASTn. The biochemical profile was assessed for catalase, hemolysis, motility, indole, ornithine, citrate utilization, acetoin production, methyl red test, nitrate reduction, esculin hydrolysis, amylase production, and carbohydrate assimilation. Probiotic potential was evaluated based on tolerance to low pH, bile salts, and antagonistic activity against microbial pathogens. The isolated strains were identified as Gram-positive bacilli, endospore formation, and they tested positive for catalase, amylase, citrate, motility, nitrate reduction, esculin hydrolysis. They assimilated glycerol, glucose, fructose, mannitol, and sucrose. The strains with the highest confidence values from MALDI-TOF (≥2.0) were strains 93 and 94, which exhibited tolerance to pH 2, 0.3% bile salts, and antagonistic effects against pathogens. The strains were identified as B. amyloliquefaciens and B. subtilis. Other studies have documented the presence of Bacillus subtilis in Tepache, but not B. amyloliquefaciens.