Probiotic efficiency of lactobacillus strains was isolated from milk sample and their antibacterial activities against human bacterial pathogens were screened in the present study. The milk samples were collected in sterile containers from different places of Tamil Nadu, India. The samples were analysed qualitatively and microbiologically. Lactobacillus was isolated from the milk samples and it was identified by biochemical tests. Methylene blue reduction test, which is colour sensitive to oxygen concentration is added to the milk. Resazurin test used to screen milk quality based either on the colour production. The total bacterial population count of milk sample was enumerated by Pour plate technique. MRS agar plates were prepared and a loopful of milk sample is taken. Quadrant streak procedure was carried out to isolate Lactobacillus. The isolated strains are identified and used to study their antagonistic efficiency against human bacterial pathogens isolated from different pathologic medium from patients diagnosed to have various wound infection at the laboratory Joys, Nagercoil, Kanyakumari (district) Tamilnadu. The probiotic action of Lactobacillus was studied against some human pathogens like Eschericia coli, Staphylococcus aureus, Enterobacter aerogenes, Streptococcus pyogenes, Pseudomonas aeruginosaand Salmonella typhimurium and it was observed that Lactobacillus showed good antagonistic effect against these pathogens.