The objective of this study was to characterize native Lactobacillus fermentum isolates for their probiotic attributes. Accordingly, 12 L. fermentum isolates selected from indigenous fermented dairy products and infant fecal samples were evaluated for their probiotic properties by in vitro and PCR methods. The cultures exhibited high tolerance to acid and bile as well as survival in simulated transit fluids (above 70 %). Cell surface hydrophobicity was in the range of 0.55-57.69 % for xylene and 0.45-77.12 % for hexadecane, whereas auto-aggregation ranged between 9 and 62 %. Isolates exhibited efficient binding to mucin and fibronectin, bile salt hydrolase activity, cholesterol assimilation (49-76 %), and radical scavenging activity (37-77 %). The isolates demonstrated antibacterial activity against Listeria monocytogenes Scott A and Micrococcus luteus ATCC 9341. Molecular fingerprinting and identification of the isolates were achieved by PCR with GTG(5) as well as 16S rRNA, phenylalanyl-tRNA synthetase alpha subunit (pheS), and RNA polymerase alpha subunit (rpoA) genes. This revealed the genomic diversity of the isolates from the two sources. Gene-specific amplification of probiotic marker genes was attained by PCR-based methods, and resultant products were sequenced. Multiple sequence alignment of the probiotic marker genes using bioinformatics revealed similarity to completely sequenced genomes of L. fermentum CECT 5716 and IFO 3956 with a few variations in mucin-binding protein gene sequences. Isolates designated as L. fermentum MCC 2759 and L. fermentum MCC 2760 showed the best probiotic attributes with high survival in simulated gastrointestinal fluids, in vitro adhesion, cholesterol reduction, and high antioxidative potential. Thus, these cultures could be potential probiotic candidates for application as functional foods.