To evaluate the phage endolysin SAL-1 as a therapeutic agent for Staphylococcus aureus infections, the in vitro and in vivo antibacterial properties of a pre-formulation containing recombinant SAL-1 as an active pharmaceutical ingredient were investigated. The stable pre-formulation (designated SAL200) uniquely included calcium ions and Poloxamer 188 as enhancing and stabilising ingredients, respectively. SAL-1 was successfully produced with no extraneous amino acids by decreasing the culture temperature and was highly purified using a two-step chromatography procedure consisting of ion exchange and hydrophobic interaction chromatography. SAL200 exhibited rapid and effective bactericidal activity against encapsulated and biofilm-forming S. aureus as well as against planktonic S. aureus cells. In addition, SAL200 demonstrated increased effectiveness in the serum environment, with a significantly reduced minimum bactericidal concentration compared with that determined in culture medium. In in vitro antibacterial tests performed against 425 clinical isolates [including 336 meticillin-resistant S. aureus (MRSA) isolates and 1 vancomycin-intermediate S. aureus isolate], collected from 421 patients and four animals, SAL200 exhibited obvious antibacterial activity against all S. aureus isolates tested. Intravenous injection of SAL200 in a mouse model of MRSA infection prolonged the viability of mice and significantly reduced bacterial counts in the bloodstream and splenic tissue. The results presented in this article strongly support SAL200 as a highly potent bactericidal agent against MRSA with an adequate pharmaceutical formulation.