Pseudomonas aeruginosa is a pathogen that can cause serious infections and usually coexists with other pathogens, such as Staphylococcus aureus. Virulence factors are important for maintaining a presence of the organisms in these multispecies environments, and DesB plays an important role in P. aeruginosa virulence. Therefore, we investigated the effect of DesB on S. aureus reduction under competitive situation. Liquid cultures of P. aeruginosa wild type (WT) and its desB mutant were spotted on agar plates containing S. aureus, and the size of the clear zones was compared. In addition, interbacterial competition between P. aeruginosa and S. aureus was observed over time during planktonic coculture. The transcriptional profiles of the WT and desB mutant were compared by qRT-PCR and microarray to determine the role of DesB in S. aureus reduction at the molecular level. As a result, the clear zone was smaller for the desB mutant than for P. aeruginosa PAO1 (WT), and in planktonic coculture, the number of S. aureus cells was reduced in the desB mutant. qRT-PCR and microarray revealed that the expression of MvfR-controlled pqsA-E and phnAB operons was significantly decreased, but the mexEF-oprN operon was highly expressed. The results indicate that intracellular levels of 4-hydroxy-2-heptylquinoline (HHQ), a ligand of MvfR, are reduced due to MexEF-OprN-mediated efflux in desB mutant, resulting in the decrease of MvfR binding to pqsA-E promoter and the reduction of 4-hydroxy-2-alkylquinolines (HAQs) synthesis. Overexpression of mexEF-oprN operon in desB mutant was phenotypically confirmed by observing significantly increased resistance to chloramphenicol. In conclusion, these results suggest that DesB plays a role in the inhibition of S. aureus growth by controlling HAQ synthesis.