Staphylococcus aureus, a prominent pathogen, is frequently encountered in clinical and food-processing settings. Given its ability to develop antimicrobial resistance, effective control strategies are required to ensure microbial safety. In this study, four lytic S. aureus phages (SapYZU10, SapYZU11, SapYZU12, and SapYZU13) were isolated from sewage samples in Yangzhou, China. Their biological characteristics and bactericidal effect against S. aureus isolates in vitro and in milk and fresh pork were evaluated. Their activities remained relatively stable under stressful conditions (-80–70°C, pH 3.0–12.0). Notably, SapYZU11 (100%, 53/53) effectively lysed all 53 S. aureus strains, followed by SapYZU12 (90.57%, 48/53), SapYZU13 (79.25%, 42/53), and SapYZU10 (71.70%, 38/53). Among the phages with short latent periods (10–20 min), SapYZU11 had a larger burst size (152.00 plaque forming units [PFU]/mL) and no genes related to antibiotic resistance and virulence. Furthermore, SapYZU11 effectively eradicated S. aureus and its cocktail (YZUsa1, YZUsa4, YZUsa12, YZUsa14, and methicillin-resistance S. aureus (MRSA) JCSC 4744) in Luria–Bertani broth and both food items. Particularly in milk, SapYZU11 with a multiplicity of infection (MOI) of 100 inhibited MRSA JCSC 4744 strain and S. aureus cocktail with maximum reduction levels of 5.03 log (Lg) colony-forming unit (CFU)/mL and 2.80 Lg CFU/mL, respectively. Conversely, contaminated pork treated with three MOIs of SapYZU11 at 25°C and 4°C resulted in reductions of 0.29–1.29 Lg CFU/mL and 0.11–0.32 Lg CFU/mL, respectively. Therefore, SapYZU11 proved as a promising biocontrol agent against S. aureus in different food production settings.
