Quorum sensing (QS) has been recognized to play an important role in many pathogenic bacteria and has become a novel target for the treatment of infectious disease. Pseudomonas aeruginosa is highly resistant to antibiotic treatment, largely due to its ability to form biofilms, and QS was found to be essential for the creation of mature, differentiated biofilms in this organism. A novel QS inhibitor, C2 (N-decanoyl-l-homoserine benzyl ester), can attenuate not only total protease and elastase activity, but also swarming motility and biofilm formation in the P. aeruginosa strain PAO1. We demonstrated that C2 showed a significant inhibitory effect on biofilm formation in a dose-dependent manner. Data from cDNA microarray showed that expression of 382 genes (similar to 6.4 %) was significantly different with C2 treatment, including downregulation of 215 genes (similar to 3.6 %) and upregulation of 167 genes (similar to 2.8 %). Real-time reverse transcription-polymerase chain reaction (RT-PCR) showed that the gene qscR, which encodes the LuxR-type receptor QscR (quorum sensing control repressor), was significantly upregulated by 375.4 % during C2 treatment. The mechanism by which C2 inhibits biofilm formation may be through repression of Las and Rhl systems by QscR. C2 was shown to reduce biofilm formation; in combination with antibiotics, it abolishes biofilm formation completely. This result may pave the way for new treatments for biofilm-related infections and may be exploited for the general prevention of biofilm formation.