Bacteria synchronize group behaviors using quorum sensing, which is advantageous during an infection to thwart immune cell attack and resist deleterious changes in the environment. InPseudomonas aeruginosa, thePseudomonasquinolone signal (Pqs) quorum-sensing system is an important component of an interconnected intercellular communication network. Two alkylquinolones, 2-heptyl-4-quinolone (HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS), activate transcriptional regulator PqsR to promote the production of quinolone signals and virulence factors. Our work focused on the most abundant quinolone produced from the Pqs system, 2,4-dihydroxyquinoline (DHQ), which was shown previously to sustain pyocyanin production and antifungal activity ofP. aeruginosa. However, little is known about how DHQ affectsP. aeruginosapathogenicity. UsingC. elegansas a model forP. aeruginosainfection, we foundpqsmutants only able to produce DHQ maintained virulence towards the nematodes similar to wild-type. In addition, DHQ-only producing mutants displayed increased colonization ofC. elegansand virulence factor production compared to a quinolone-null strain. DHQ also bound to PqsR and activated the transcription ofpqsoperon. More importantly, high extracellular concentration of DHQ was maintained in both aerobic and anaerobic growth. High levels of DHQ were also detected in the sputum samples of cystic fibrosis patients. Taken together, our findings suggest DHQ may play an important role in sustainingP. aeruginosapathogenicity under oxygen-limiting conditions.