Bacteriophage (phage) therapy using whole phage or phage-lytic components has emerged as a promising antibacetrial strategy as an adjunct to the use of antibiotic drugs. We recently purified 16 independent isolates of Staphylococcus bacteria representing seven different species by surveying local community wastewater and discovered a bacteriophage with unusual structural properties that include a flexible and contractile tail. The phage, JS1, was isolated using Staphylococcus xylosus as a host and was subsequently shown to be capable of infecting diverse species of Staphylococcus, including a range of clinical Staphylococcus aureus strains. These bacteria are Gram-positive, meaning that they have an expansive cell wall made of peptidoglycan surrounding their single cytoplasmic membrane. Following genome sequencing and annotation three JS1 proteins were identified as putative hydrolases. Analysis of high-confidence structural predictions revealed catalytic and cell wall binding domains characteristic of hydrolase families associated with bacterial cell wall hydrolysis. One of the hydrolases, JS1_224, was biochemically characterized and exhibited a broader host range against staphylococcal strains than its parent phage (100% vs. 65.9%). Treatment with JS1_224 caused a ~100-fold reduction in viable colony forming units (CFU/mL) of Staphylococcus in vitro. Scanning electron microscopy revealed cell wall perforation following a short duration of treatment with JS1_224, and severe defects in cell morphology including lysis with prolonged treatment. Overall, the data in the study highlights the advantages of phage-derived hydrolases in combatting gram-positive Staphylococcus in vitro.