Abstract: OBJECTIVE To investigate the antagonistic activity of Serratia marcescens against Staphylococcus aureus. METHODS Clinical isolates from the Second Affiliated Hospital of Anhui Medical University from Apr. 2022 to Jun. 2023 were screened for S. marcescens with antagonistic activity against S. aureus with the spread-spot inoculation method. The isolates were classified into non-pigmented and pigmented S. marcescens based on colony color. Species identification was performed with the VITEK 2 GN identification card and Microflex LT matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) analyzer, and a phylogenetic tree was constructed through 16s rRNA sequence alignment. The antagonistic activity of S. marcescens against methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) at different temperatures was evaluated through the Oxford cup method. The hemolytic toxicity of the sterile fermentation supernatant of S. marcescens on human red blood cells was detected with the microplate method. RESULTS A total of 8 S. marcescens strains with antagonistic activity against MRSA and MSSA were screened. These strains had neat colony edges and smooth surfaces, and all carried the pswP gene. Three pigmented strains carried the swrW gene, while 3 out of 5 non-pigmented strains carried the swrA gene. Both pigment and temperature affected the antagonistic activity of S. marcescens, with an interactive effect. Under conditions of 25, 30 and 35 ℃, non-pigmented S. marcescens exhibited stronger antagonistic activity than pigmented S. marcescens. At 40 ℃, neither showed antagonistic activity, and pigmented S. marcescens failed to synthesize pigment. The hemolysis rate of human red blood cells by the sterile fermentation supernatant of non-pigmented S. marcescens was <2.5%, which was lower than that of pigmented S. marcescens (P<0.05). CONCLUSIONS The non-pigmented S. marcescens included in this study demonstrates significant antagonistic activity against S. aureus. The non-prodigiosin, non-surfactant antimicrobial active components secreted by these strains have the potential to serve as lead compounds for the development of novel antimicrobial drugs.