Emergence of multidrug resistant strains has created serious problem for safe eradication of Staphylococcus aureus infections. Therefore, there is an urgent need to develop novel antibacterial agents to control this pathogen. Bacteriophages kill bacteria irrespective of their antibiotic sensitivity and thus they can be used as potent prophylactic/therapeutic agent to treat such infections. Here, we report isolation of broad host range anti-staphylococcal lytic bacteriophage P-27/HP from sewage water. This phage was able to inhibit 17 of 28 (60%) human disease associated S. aureus isolates. In vitro studies revealed its strong lytic efficacy to diminish S. aureus 27/HP population (c.f.u.) by more than 5.0 logs (P < 0.0001) (equivalent to 99.99%) in 3 h at 0.01 MOI. In vivo lytic efficacy analysis showed that a single subcutaneous injection of phage P-27/HP (10(7) p.f.u.) was sufficient to protect S. aureus 27/HP infected (5 x 10(8) c.f.u.) mice from bacteremia and subsequent death. A considerable decline of more than 6 logs (99.9%) in splenic S. aureus 27/HP c.f.u. count was noted at the 3 days of phage treatment. In conclusion, our results suggest that phage P-27/HP is polyvalent in nature and has high-lytic potential towards S. aureus, thus, a therapy employing this phage would be efficacious to control S. aureus infections.