Bifidobacterium strains inhibit growth or cell adhesion of pathogenic bacteria and against multidrug-resistant bacteria. Furthermore, when combined with certain antibiotics, probiotics can boost their antibacterial activity. The objective of present report was to evaluate the antibacterial effect of Bifidobacterium spp. In the first part of the investigation, we evaluated the antibacterial activities of viable and inactivated cells, from a total of 12 Bifidobacterium species, on the growth of three different common pathogens associated with multi-drug resistance such as Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and Eenterococcus faecalis. Also, eight strains of sonication-inactivated Bifidobacteria exhibited antibacterial activity against S. aureus and P. aeruginosa. Additionally, eight viable Bifidobacterium strains exhibited antibacterial activity on the growth of pathogenic species. Therefore, we observed the antimicrobial activity, of Bifidobacteria against pathogenic bacteria, on the solid medium utilizing the agar well diffusion method. Some Bifidobacterium supernatants such as B. longum and B. pseudocatenulatum, showed synergism with the antibacterial activity of antibiotics. The results exhort that Bifidobacteria could be employed as an effective control for nosocomial pathogenic bacteria, and reduce the risk of the development of S. aureus and P. aeruginosa infection. The present authors propose that Bifidobacteria might be a useful probiotic microorganism, for combining modality with antibiotics, without adverse effects.