Methicillin-resistant Staphylococcus aureus (MRSA) can cause a wide range of infections from mild to life-threatening conditions. Its enhanced antibiotic resistance often leads to therapeutic failures and therefore alternative eradication methods must be considered. Potential candidates to control MRSA infections are bacteriophages and their lytic enzymes, lysins. In this study, we isolated a bacteriophage against a nosocomial MRSA strain belonging to the ST45 epidemiologic group. The phage belonging to CaudoviralesSiphoviridae, showed a narrow host range and stable lytic activity without the emergence of resistant MRSA clones. Phylogenetic analysis showed that the newly isolated Staphylococcus phage R4 belongs to the Triavirus genus in Siphoviridae family. Genetic analysis of the 45 kb sequence of R4 revealed 69 ORFs. No remnants of mobile genetic elements and traces of truncated genes were observed. We have localized the lysin (N-acetylmuramoyl-L-alanine amidase) gene of the new phage that was amplified, cloned, expressed, and purified. Its activity was verified by zymogram analysis. Our findings could potentially be used to develop specific anti-MRSA bacteriophage- and phage lysin-based therapeutic strategies against major clonal lineages and serotypes.