For a study, it was determined that because of the potential health benefits of probiotic bacteria, new microbial strains were isolated for use in food products. The “generally recognized as safe” (GRAS) designation of freshly isolated potential probiotic organisms was not automatically shared by traditional lactic acid bacteria (LAB). New isolates must be tested for safety before being included in food items. The researchers sought to characterize LAB isolates from a newborn infant’s stool in vitro and assess their safety and probiotic potential. Lactobacillus gasseri was discovered in 30 colonies using 16S rDNA sequencing. Using the restriction enzymes SmaI and Apa I, Pulsed Field Gel Electrophoresis indicated that 29 of the L. gasseri isolates were essentially identical, but one isolate had a different DNA fingerprint. All 30 L. gasseri strains were tested for antibiotic resistance, bile tolerance, hemolytic activity, and pathogen antagonism. All 30 strains had 3 plasmids, with 1 strain having a 4th plasmid encoding a potential multidrug resistance transporter protein and showing significant tolerance to 0.5% bile (LmrB). Aside from acid inhibition, there was no evidence of hemolytic action or antagonism. The 3 isolates, UFVCC1083, 1091, and 1112, showed significant resistance to simulated small intestinal and gastric fluids and adhered to mucin and two intestinal epithelial cell lines, Caco-2 and HT-29, in vitro. Finally, it was discovered and evaluated that newly obtained L. gasseri strains from a breastfed infant’s feces as possible probiotic candidates for use in Brazilian human milk banks.