ABSTRACTStaphylococcus aureusis an important human pathogen and brings about many community-acquired, hospital-acquired, and biofilm-associated infections worldwide. It tends to form biofilms, triggering the release of toxins and initiating resistance mechanisms. As a result of the development ofS. aureustolerance to antibiotics, there are few drugs can availably control biofilm-associated infections. In this study, we synthesized a novel small-molecule compound CY-158-11 (C22H14Cl2NO2Se2) and proved its inhibitory effect on the biofilm formation ofS. aureusat a subinhibitory concentration (1/8 MIC). The subinhibitory concentration of CY-158-11 not only did not affect the growth of bacteria but also had no toxicity to A549 cells orG. mellonella. Total biofilm biomass was investigated by crystal violet staining, and the results were confirmed by SYTO 9 and PI staining through confocal laser scanning microscopy. Moreover, CY-158-11 effectively prevented initial attachment and repressed the production of PIA instead of autolysis. RT-qPCR analysis also exhibited significant suppression of the genes involved in biofilm formation. Taken together, CY-158-11 exerted its inhibitory effects against the biofilm formation inS. aureusby inhibiting cell adhesion and the expression oficaArelated to PIA production.IMPORTANCEMost bacteria exist in the form of biofilms, often strongly adherent to various surfaces, causing bacterial resistance and chronic infections. In general, antibacterial drugs are not effective against biofilms. The small-molecule compound CY-158-11 inhibited the biofilm formation ofS. aureusat a subinhibitory concentration. By hindering adhesion and PIA-mediated biofilm formation, CY-158-11 exhibits antibiofilm activity towardS. aureus. These findings point to a novel therapeutic agent for combating intractableS. aureus-biofilm-related infections.