Quorum-sensing is a phenomenon of intercellular messages in microorganisms through secretion of small signaling molecules, which spread between cells, facilitating communication, and regulating virulence factors production. Quorum-sensing system disruption in Pseudomonas aeruginosa through identifying new inhibitors is a promising approach. Herein, we test the possible quorum-sensing inhibitory effect of esomeprazole, a proton pump inhibitor, on the standard strain Pseudomonas aeruginosa PAO1. The production of the quorum-sensing controlled violacein pigment in the biosensor microorganism Chromobacterium violaceum- CV026 was significantly decreased by using 1/4 minimal inhibitory concentration of esomeprazole. For Pseudomonas aeruginosa PAO1, such concentration of esomeprazole inhibited the swarming motility and reduced the production of the quorum-sensing dependent virulence factors proteases, elastase, pyoverdin, and pyocyanin. qRT-PCR confirmed that esomeprazole significantly decreased the relative expression levels of the quorum-sensing regulated genes LasI, LasR, RhlI, RhlR, PqsA, and PqsR which control virulence factors production. Docking study revealed potential strong binding capability of esomeprazole with LasR, RhlR and PqsR receptors. To summarize, this study showed that esomeprazole could be used as a valuable quorum-sensing inhibitor that can help develop a new approach for identifying alternative solutions to treat pseudomonal infections that may not respond to traditional antibiotics.