Background: The ica gene of Staphylococcus aureus (S. aureus) plays a vital role in its growth and biofilm formation. Among them, IcaA and IcaB are critical proteins for synthesizing extracellular polysaccharides and biofilms in S. aureus. To investigate whether the formation of S. aureus biofilms can be inhibited through the IcaA and IcaB proteins by the presence of linezolid. Methods: The icaA and icaB genes of S. aureus ATCC 25923 were silenced by homologous recombination. The critical roles of icaA and icaB in S. aureus were analysed by observing the growth curve and biofilm formation after linezolid treatment. Then, the effect of linezolid on the morphology of S. aureus was observed by scanning electron microscopy. Finally, the potential binding ability of linezolid to Ica proteins was predicted by molecular docking. Results: The icaA- and icaB-silenced strains were successfully constructed, and the sensitivity of S. aureus to linezolid was decreased after icaA and icaB silencing. Scanning electron microscopy showed that linezolid caused invagination of the S. aureus surface and reduced the production of biofilms. Molecular docking results showed that linezolid could bind to IcaA and IcaB proteins. Conclusion: IcaA and IcaB are potential targets of linezolid in inhibiting the biofilm formation of S. aureus (ATCC 25923).  Kulcsszavak: Staphylococcus aureus, biofilm, icaA, icaB, linezolid