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Abstract Staphylococcus aureus (S. aureus) is known as a food-borne pathogenic bacteria that is difficult to deal with, especially in a biofilm. An appropriate and effective agent against S. aureus is urgently needed, especially due to the high pathogenicity of S. aureus within biofilms. This paper aims to synthesize Ag 3 PW 12 O 40 nanoparticles (AgWPA-NPs) and investigate their antimicrobial activity and mechanism against S. aureus. N-phenyl-1-naphthylamine (NPN) uptake and membrane potential assays showed that AgWPA-NPs damaged the structure of the cell membrane. Furthermore, the results of crystal violet staining and fluorescence microscopy indicated that AgWPA-NPs were remarkably efficient at suppressing the formation of S. aureus biofilms. Furthermore, real-time PCR analysis showed that the expression of the biofilm-related genes, icaA , sarA and cidA , was downregulated. Taken together, these results suggest that AgWPA-NPs have the potential to be an anti- S. aureus biofilm agent. Highlights • AgWPA-NPs damaged the structure of S. aureus cell membrane. • AgWPA-NPs inhibited growth of S. aureus in biofilm and eliminated S. aureus biofilm. • AgWPA-NPs inhibited the biofilm-related genes and expression of SEA and SEB. [ABSTRACT FROM AUTHOR]