Yunnan Baiyao is a famous Chinese medicine that has long been directly applied to wounds to reduce bleeding, pain, and swelling without causing infection. However, little is known about its ability to prevent infection. The present study aimed to assess in vitro the anti-virulence activity of an aqueous extract of Yunnan Baiyao (YBX) using Pseudomonas aeruginosa as a pathogenic model. We found that a sub-MIC (2.5 mg/ml) of YBX can efficiently interfere with the quorum-sensing (QS) signaling circuit. Real-time polymerase chain reaction analysis showed that a sub-MIC of YBX down-regulated the transcriptions of lasR, lasI, rhlR, and rhlI, which resulted in global attenuation of QS-regulated virulence activities, such as biofilm formation, and secretion of LasA protease, LasB elastase and pyocyanin. Further, YBX reduced the motility of P. aeruginosa related to QS, and impaired the formation of biofilms. These results suggest that YBX may possess global inhibitory activity against the virulence of P. aeruginosa and that YBX may also exhibit antimicrobial activity in vivo. The present study suggests that Yunnan Baiyao represents a potential source for isolating novel, safe, and efficacious antimicrobial agents.