Staphylococcus aureus is one of the leading pathogens responsible for antibiotic resistance-related deaths. S. aureus can secrete a variety of exotoxins, and staphylococcal biofilms play an important role in antibiotic resistance and persistence of chronic infections. Here, we investigated the inhibitory effects of 18 hydroquinones on biofilm formation and virulence factor production by S. aureus. It was found that 2,5-bis(1,1,3,3-tetramethylbutyl) hydroquinone (TBHQ) at 1 ㎍/mL efficiently inhibits biofilm formation by two methicillin-sensitive and two methicillin-resistant S. aureus strains with MICs of 5 ㎍/mL, whereas the backbone compound hydroquinone did not (MIC > 400 ㎍/mL). In addition, 2,3-dimethylhydroquinone and tert-butylhydroquinone at 50 ㎍/mL also exhibited antibiofilm activity. TBHQ at 1 ㎍/mL significantly decreased the hemolytic effect and lipase production by S. aureus, and at 5 - 50 ㎍/mL was non-toxic to the nematode Caenorhabditis elegans and did not adversely affect Brassica rapa seed germination or growth. Transcriptional analyses showed that TBHQ suppressed the expression of RNAIII (effector of quorum sensing). These results suggest that hydroquinone, especially TBHQ, is potentially useful for inhibiting S. aureus biofilm formation and virulence.