PURPOSE: Quorum sensing (QS) system is crucial for biofilm formation, virulence and survival of Pseudomonas aeruginosa (P. aeruginosa). With the rise of antibiotic-resistant bacteria, QS inhibitors (QSIs) are seen as potential agents to mitigate infections. Plant-based natural products are rich sources of anti-QS compounds. Viola philippica Cav. (Violaceae) is traditionally used to treat inflammatory diseases, respiratory diseases and anti-microbials. However, its anti-QS effects against P. aeruginosa are poorly understood. We aimed to reveal the effects of scopoletin and scoparone from V. philippica Cav. extract (VPE) on QS-related virulence phenotypes and genes in P. aeruginosa PAO1. MATERIALS AND METHODS: Compounds in VPE were identified using ultrahigh-performance liquid chromatography coupled with Q-exactive hybrid quadrupole-Orbitrap mass spectrometry (UHPLC-Q Exactive-Orbitrap MS). Virulence factors of PAO1, including pyocyanin, rhamnolipids, elastase, hemolysin, swarming and swimming motility, and biofilm formation, were evaluated. Real-time reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect virulence-related genes. Bioassays for the QS inhibitory activity of VPE and its compounds were performed using Chromobacterium violaceum (C. violaceum) CV026. RESULTS: Sub-minimum inhibitory concentrations (sub-MICs) of VPE inhibited QS-related virulence factors in PAO1, including pyocyanin, rhamnolipids, biofilms, and swarming motility. Scopoletin and scoparone, two coumarin derivatives identified in VPE using UHPLC-Q Exactive-Orbitrap MS, also showed anti-QS activity against PAO1. These compounds reduced pyocyanin, rhamnolipid, and hemolysin production, elastase activity, biofilm formation and swarming motility. Further RT-qPCR assays revealed that scopoletin and scoparone universally suppress QS-associated genes (lasI, rhlI and pqsA) in PAO1, primarily during the early and middle stages of growth. CONCLUSION: Scopoletin and scoparone, two previously uncharacterized coumarin derivatives from VPE, were identified as novel QSIs. They are responsible for VPE's effect on weakening QS-controlled virulence of PAO1 by inhibiting QS-related genes. This study provides a laboratory basis for using scopoletin and scoparone as promising anti-virulence candidates for adjuvant therapy against P. aeruginosa infection.