Pseudomonas aeruginosa quorum sensing (QS) system is a cell to cell signaling mechanism that regulates virulence factors and pathogenicity. Therefore, the QS system in P. aeruginosa may be an important target for pharmacological intervention. The present study aimed to investigate the effects of sub-MIC concentrations of (S,E)-2-hydroxy-N-(3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl)propane hydrazide (pyridoxal lactohydrazone) against P. aeruginosa QS related virulence factors. We investigated the effect of sub-MIC concentrations of chiral pyridoxal lactohydrazone, which formed by the reaction of chiral lactic acid hydrazide and pyridoxal (one form of Vitamin B6) as bioactive reagents, on virulence factors. Treated PAO1 cultures in the presence of tested compound at 1/4 and 1/16 MIC (32 and 8 μg/mL respectively) showed significant inhibition of virulence factors including motility, alginate and pyocyanin production and susceptibility to H2O2 (P < 0.001). Also, the pyridoxal lactohydrazone showed anti-QS activity in Chromobacterium violaceum CV026 biosensor bioassay. Because of quorum sensing is a promising target for anti-virulence therapy and also important role of LasR regulatory protein in the initiation of P. aeruginosa QS system, we carried out molecular docking for understanding the interactions of pyridoxal lactohydrazone with the LasR receptor. The results of docking study suggested that the pyridoxal lactohydrazone has potential to inhibit the LasR protein. The results indicated that sub-MIC concentrations of this compound exhibited inhibitory effect on P. aeruginosa QS related virulence factors.