A novel bacteriophage, phi MR25, was isolated from a lysogenic Staphylococcus aureus strain by mitomycin C induction. Its biological features were analyzed in comparison with phi MR11, which was described previously as a prototype therapeutic phage. phi MR25 is morphologically similar to phi MR11 (morphotype B1 of family Myoviridae) but has a broader host range than phi MR11 on S. aureus strains. phi MR25 can also multiply on S. aureus lysogens of phi MR11. Its DNA is 44,342 bp in size, is predicted to include 70 open reading frames, and does not contain genes related to toxin or drug resistance. The lysogenic module and most of the putative virion protein genes are completely different from those of phi MR11. In spite of their genetic diversity, intraperitoneal administration of phi MR25 rescued mice inoculated with a lethal dose of S. aureus, as was the case for phi MR11. These results suggest that phi MR25 could be another candidate phage to treat S. aureus infection.