Human milk is a complex biological fluid, and a source of nutrients and probiotics which plays a vital role in the growth and development of infants. Probiotics are helpful bacteria which are good for human health. The primary bacterial genera which are gaining status as probiotics are Lactobacillus, Lactococcus, and Bifidobacterium. The present work aimed to isolate, identify, and determine the probiotic potential of Lactobacillus bacteria from human milk. A total of 70 samples of human milk were collected from different lactating mothers. The milk samples were inoculated on the De Man, Rogosa, and Sharpe (MRS) agar plates to observe the growth of Lactobacillus bacteria. The bacteria were identified based on their morphology, culture characteristics, and biochemical properties. Isolated bacteria were evaluated for probiotic properties in which, tolerance to acidic pH, bile salts, and gastric juice as well as antibacterial activity and antibiotic susceptibility were determined. Out of the 70 milk samples, 57 were positive for Lactobacillus. Out of the positive sample, 10.5% of the samples tolerated acidic pH and high bile salt concentration, but a significant difference was obtained for gastric juice. In the antibacterial activity, Pseudomonas showed no action against Lactobacillus. In antibiotic susceptibility, the test isolates were resistant to penicillin. The present work proved the presence of beneficial bacteria in the human milk. Isolated Lactobacillus exhibited significant antibacterial activity against pathogenic bacteria, and tolerance to acidic pH, bile salt, and gastric juice. Therefore, human milk could be a good source of probiotics for infants.