The rise in multidrug resistance and strong biofilm-forming ability of Staphylococcus aureus has led to significant public health concerns. Phage or phage-derived components, such as depolymerase or endolysin, have been considered as potential alternatives to antibiotics for combating antibiotic-resistant bacterial infections. In this study, we cloned and expressed a Staphylococcus aureus phage endolysin, LysP4, and identified its lytic activity. The bactericidal effect of LysP4 was more pronounced against planktonic cells in the logarithmic phase compared to those in the stationary phase. LysP4 reduces bacterial counts by 3 log CFU/mL in 60 min and about 2 log CFU/mL during the stationary phase. LysP4 exhibited optimal lytic activity at pH 5.0-7.0 and remained stable across a temperature range of 16 to 40 °C, with maximal activity observed at 37 °C. LysP4 effectively targets 31 of 38 Staphylococcus strains and successfully eliminates biofilms, reducing bacterial counts by 4 log CFU/mL when combined with vancomycin. Notably, LysP4 demonstrated no hemolytic effects on human red blood cells and no toxic effects on embryonic kidney cells or lung cancer cells. Based on these findings, we believe that LysP4 holds promise as a biological control agent against Staphylococcus infections.© 2025. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.