Bacteriocins are ribosomal-synthesized peptides produced by Lactic acid bacteria which can kill other bacteria. The present study aimed to study the effect of bacteriocin produced by three different Lactobacillus strains, (Lactobacillus helveticus, Lactobacillus rhamnousus and Lactobacillus acidophilus) as a natural bio preservative in Domiati cheese. The chemical composition, antioxidant and antibacterial activity was determined during room temperature storage for 3 months which affected the quality of cheese and accelerated its spoilage. The highest antioxidant and antibacterial activity produced by Lb. helveticus followed by Lb. rhamnousus. The moisture content of all cheese treatments showed a significantly decreased (p < 0.05) during ripening due to the biochemical changes after 3 months. All cheese treatments exhibited higher acidity than the control. Fat content in all treatments significantly increased throughout the ripening period due to the decrease in the moisture content and there were no significant differences between treatments. Water-soluble Nitrogen (WSN) content significantly increased by adding Lactobacillus strains after 3months of storage. Hence, the purified Cell-Free Supernatant (CFS) from Lb.helveticus at a concentration of 2.5µg/ml, which was more effective than other strains, would be used as a protective culture to delay the growth of pathogenic bacteria, particularly S. typhimurium and L. monocytogenes in Domiati cheese.