Context . Pseudomonas aeruginosa (P aeruginosa) is an opportunistic and nosocomial, Gram-negative pathogen that is characteristic of quorum sensing (QS) modulation. QS inhibition (QSI) is considered to be a new approach to antimicrobial treatment that may reduce issues of antibiotic resistance. Traditional Chinese herbs have been recognized as a source of QSI. Objective . This study was intended to investigate whether the Huang Lian Jie Du decoction (HLJJD) possesses the ability to produce anti-QS activity. Design . P aeruginosa PAO(1) was cultivated in the presence and absence of HLJDD or of the aqueous extracts of each of its 4 constituent herbs: Rhizoma coptidis (RC), Radix scutellariae (RS), Cortex phellodendri (CP), and Fructus gardeniae jasminoidis (FGJ). The minimum inhibitory concentrations (MICs) were found for the HLJDD or its 4 constituents through testing each for evidence of antibacterial activity at 3.13, 6.25, 2.5, 25, 50, 100, 200, and 400 mg/mL. Outcome Measures . QS-dependent virulence factors were analyzed at subminimum MICs (sub-MICs) for the HLJDD and its constituent herbs. The mRNA of the QS genes was also assessed by a quantitative real-time polymerase chain reaction. Results . The HLJDD exhibited the lowest MIC of 100 mg/mL, compared with MICs of 200 mg/mL for the RC and RS, 400 mg/mL for the CP, and more than 400 mg/mL for the FGJ. At the sub-MIC of 50 mg/mL for HLJDD, the results showed a significant reduction in pyocyanin pigment, elastolytic activity, proteolytic activity, biofilm formation, and bacterial motilities in the PAO(1), without inhibiting the bacterial growth. The mRNA level of the P aeruginosa elastase gene (lasB) was also significantly reduced. The mRNA analysis also showed an increasing tendency for overexpressed QS genes when observed at sub-MICs. That inhibitory tendency was also observed in the sub-MICs of the constituent herbs in different degrees. Conclusions . The HLJDD showed the synergism of its 4 constituents against the growth of P aeruginosa and the QS-dependent virulence factors. Its anti-QS activity may interrupt the QS domination of the virulence factors of P aeruginosa downstream, leading to the overexpressed QS genes.