A study was performed to identify and characterize 99 indigenous strains isolated from human breast milk (n=29) and fecal samples (n=70) in Ghana, using methods ranging from plating growth tests, conforming to (NCCLS) standards and presumptive analysis at species level using MALDI-TOF MS, prior to validation through qPCR techniques. Antimicrobial activities of the isolated strains were performed by the agar well diffusion assay. Gastrointestinal tolerance and ability to grow biofilms in vitro using 3D Alvatex platform were determined. Results indicate that out of 99 samples, 25 were gram-positive, catalase-negative rods, 80% of which were Lactobacillus strains. More than one-third of the identified strains were L. fermentum followed by L. plantarum, L. rhamnosus, L. salivarius, L. reuteri along with Enterococcus faecium, Weissella spp. and Pediococcus spp. Among the 25 isolates, 9 had activity against both gram-negative and gram-positive tested pathogens including Staphylococcus aureus, Escherichia coli ATCC 25922, E. coli BAA-2471, Salmonella typhi, and Pseudomonas aeruginosa. The indigenous microbiota proved to be most effective antimicrobial to local pathogens over nonindigenous pathogens. The data also showed maintained cell viability for all studied isolate at pH 2, 3 and 6 followed by a successful growth of the co-cultured biofilm in a 3D Alvatex platform.