Abstract: Lactic Acid Bacteria (LAB) commonly used in food as starter cultures are known to produce antimicrobial substances such as bacteriocins and have great potential as food bio-preservatives. Six L. acidophilus isolated from traditional yoghurt, were screened for the production of antimicrobial substances. Lactobacillus acidophilus isolated from bio-yoghurt showed the broadest spectrum of antimicrobial activity was selected for further characterization. The growth of isolates was investigated in MRS medium containing 1-2% glucose at pH 6.5-7. Purification of the active compound was achieved after gel filtration and ion exchange chromatography. As revealed by SDS-PAGE, active fractions were relatively homogeneous, showing a protein with molecular mass of 30 kDa. It was stable to heat (100°C for 15 min). The bacteriocin-like substance was active against the gram positive bacteria Bacillus cereus and Staphylococcus aureus and against Pseudomonas aeroginosa and Klepsiella pneumona. The antimicrobial activity against P. aeroginosa was stronger than S. aureus. These antibacterial activities of bacteriocins-like substance were determined under absence of organic acids and hydrogen peroxide in medium.