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Hydroquinine is an organic alkaloid compound that exhibits antimicrobial activity against several bacterial strains including strains of both drug-sensitive and multidrug-resistant <i>P.</i> <i>aeruginosa</i>. Despite this, the effects of hydroquinine on virulence factors in <i>P. aeruginosa</i> have not yet been characterized. We therefore aimed to uncover the mechanism of <i>P. aeruginosa</i> hydroquinine-sensitivity using high-throughput transcriptomic analysis. We further confirmed whether hydroquinine inhibits specific virulence factors using RT-qPCR and phenotypic analysis. At half the minimum inhibitory concentration (MIC) of hydroquinine (1.250 mg/mL), 254 genes were differentially expressed (97 downregulated and 157 upregulated). We found that flagellar-related genes were downregulated by between −2.93 and −2.18 Log₂-fold change. These genes were consistent with the analysis of gene ontology and KEGG pathway. Further validation by RT-qPCR showed that hydroquinine significantly suppressed expression of the flagellar-related genes. By analyzing cellular phenotypes, <i>P. aeruginosa</i> treated with ½MIC of hydroquinine exhibited inhibition of motility (30–54% reduction) and pyocyanin production (~25–27% reduction) and impaired biofilm formation (~57–87% reduction). These findings suggest that hydroquinine possesses anti-virulence factors, through diminishing flagellar, pyocyanin and biofilm formation.