Background: Bacteria have always been present in our food. Some of them, such as lactic acid bacteria, are useful, while others, such as pathogenic bacteria, are not, because of the many problems of food poisoning and illness that they cause, as well as the multiple spoilage of foodstuffs that they provoke. As a result, food safety is a constant concern. Methods: Three experiment (Bbv1, Bbv2 and BLE) confirmed by the F6PPK activity and carbohydrates fermentation and one reference bifidobacterial (BLR) strains were examined for there antimicrobial activity against S. aureus 49444 in broth and agar by cellular and supernatant contact. Results: In associated cultures, Bifidobateria strains exhibit a strong antagonistic activity towards S. aureus 49444 by agar spot contact (plus to 8mm for all strains) or in broth contact (total inhibitory after 24 hours of contact). The bifidobacterial supernatant contact by agar spot method shows a good inhibitory activity (the inhibitory zones exceed 8mm). The supernatant contact results in broth medium demonstrated a significant reduction of S. aureus level (plus to 61% after 48 hour) with bifidobacterial supernatant contact incubated at 6°C. At 37°C a great reduction of S. aureus population was noticed to a total destroy (after 12 hour for Bbv1, Bbv2 and 24 hour of contact for BLE and BLR). The bifidobacterial antimicrobial activity of supernatant was resistant to different organic solvents. Conclusion: The results of this study give us a clear idea of the effectiveness of the Bifidobateria strains tested against S. aureus, and constitute a promising means of biological control for future tests against pathogenic microorganisms.