Background and Aim: Many local dairy products in Iran contain lactic acid bacteria. The aim of this study was to isolate and identify Lactobacillus brevis from the cottage cheese made in Bazoft city using biochemical and molecular methods and investigate its antimicrobial properties in vitro. Materials and Methods:In this study, ten local Bazoft city cheese samples were collected. After transferring to the Lab, serial dilution was obtained at 10 -6 CFU/mL and cultured in MRS medium. Then bacilli and gram-positive bacteria were isolated and identified by biochemical methods. The identified Lactobacillus brevis genome was extracted and then amplified using polymerase chain reaction (PCR) to confirm the isolated strain. Finally, the PCR product was sent to Sinaclon for sequencing. The antimicrobial properties of Lactobacillus brevis supernatant were evaluated using the disk and well diffusion method against Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa, and Enterococcus faecalis. Antibiotic susceptibility was also assessed by the CLSI standard method.Results: Six species of Lactobacillus were identified from cottage cheese samples. Selected L. brevis showed good antimicrobial potency against five pathogenic bacteria. It showed the highest inhibitory effect against S. typhimurium during the well diffusion method with a mean diameter of growth inhibition zone of 19 mm and the least inhibitory effect against E. faecalis during the disc method with a growth inhibition zone diameter of 9 mm. It was also resistant to the antibiotics Vancomycin and Gentamicin, intermediate susceptible to Kanamycin and Nalidixic acid, and susceptible to Erythromycin, Tetracycline, Ampicillin, and Chloramphenicol.Conclusion: Isolated L. brevis strain from the local Bazoft cheese has a good antimicrobial ability. By performing extra in vitro and in vivo tests, this native strain can be used more in medicines and the food industry.