The demand of functional foods is on the rise and researchers are trying to develop nutritious dairy products from well characterized bacterial strains. Lactobacillus is considered to be important bacterial species, which have been recognized as probiotics for providing host health through the improvement in the gut microbiota. The objective of present study was to isolate and molecular characterize the Lactobacillus fermentum from Nilli Ravi buffalo milk for its potential use as probiotic like fermented milk. Fifteen (15) L. fermentum were isolated and studied for acid and bile salt tolerance. Out of fifteen strains, only 4 strains (NMCC-17, NMCC-2, NMCC-28, and NMCC-14) were selected based on their maximum acid and bile salt resistance. These strains were used for in vitro study for their probiotic potential based on lysozymes tolerance, cholesterol assimilation, autoaggregation, hydrophobicity, lipolytic, proteolytic, amylolytic, beta-galactosidase test, hemolytic activity and exopolysaccharide production. NMCC-14 and NMCC-17 strains showed maximum probiotic potential. These isolates were identified as L. fermentum (MK611941 and MK614164) by 16S rRNA gene sequencing. Fermented milk sample inoculated with NMCC-14, and NMCC-17 showed decreased in live cfu/g at 4 o C at day 25. Bacterial count persistence is more than 7cfu /ml which is good sign of health benefit requirement