As antibiotic resistance escalates into a global health crisis, novel therapeutic approaches against infectious diseases are in urgent need. Pseudomonas aeruginosa, an adaptable opportunistic pathogen, poses substantial challenges in treating a range of infections. The quorum-sensing (QS) system plays a pivotal role in orchestrating the production of a large set of virulence factors in a cell density-dependent manner, and the anti-virulence strategy targeting QS may show huge potential. Here, we present a comprehensive investigation into the potential of the synthesized compound 3-(benzo[d][1,3]dioxol-4-yl)oxazolidin-2-one (OZDO, C10H9NO4) as a QS inhibitor to curb the virulence of P. aeruginosa. By employing an integrated approach encompassing in silico screening, in vitro and in vivo functional identification, we elucidated the multifaceted effects of OZDO. Molecular docking predicted that OZDO interfered with three core regulatory proteins of P. aeruginosa QS system. Notably, OZDO exhibited significant inhibition on the production of pyocyanin, rhamnolipid and extracellular proteases, biofilm formation, and cell motilities of P. aeruginosa. Transcriptomic analysis and quantitative real-time PCR displayed the down-regulation of QS-controlled genes in OZDO-treated PAO1, reaffirming the QS-inhibition activity of OZDO. In vivo assessments using a Caenorhabditis elegans-infection model demonstrated OZDO mitigated P. aeruginosa pathogenicity, particularly against the hypervirulent strain PA14. Moreover, OZDO in combination with polymyxin B and aztreonam presented a promising avenue for innovative anti-infective therapy. Our study sheds light on the multifaceted potential of OZDO as an anti-virulence agent and its significance in combating P. aeruginosa-associated infections.