Objective To explore the antimicrobial activity of the bacteriocin (tentatively named garvicin LG6) secreted by Lactococcus garvieae (L. garvieae) SHAMU-LG6 against Staphylococcus aureus (S. aureus) of different hemolytic phenotypes. Methods S. aureus isolated from clinical patients in a hospital of Anhui from 2021 to 2023 were collected. The hemolytic phenotypes of S. aureus were detected by three-point inoculation method. S. aureus of different hemolytic phenotypes were further categorized into methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) according to antimicrobial susceptibility testing results. The antagonistic activity of L. garvieae SHAMU-LG6 against S. aureus of different hemolytic phenotypes was assayed by Oxford cup me-thod. The whole-genome sequencing of L. garvieae SHAMU-LG6 was performed. Biosynthetic gene cluster of garvicin LG6 was searched by online databases antiSMASH 7.0 and BAGEL4. Through macroporous resin adsorption, ethanol gradient elution, rotary evaporation, and dried material reconstitution, antimicrobial activity of garvicin LG6 crude extract against S. aureus was detected by the inhibitory testing of Oxford cup method. Results L. garvieae SHAMU-LG6 could significantly antagonize MSSA and MRSA of different hemolytic phenotypes. Biosynthetic gene cluster of garvicin LG6 was present on the chromosomal genome of L. garvieae SHAMU-LG6. The antimicrobial activity of garvicin LG6 secreted by a single colony or 6 mL fermentation fluid of L. garvieae SHAMU-LG6 were at least equal to that of antibiotic disc of 30 μg cefoxitin. Conclusion Garvicin LG6 can efficiently kill MSSA and MRSA of different hemolytic phenotypes, and has the potential to be developed into a novel antimicrobial agent, which has great prospects for clinical application.