Pseudomonas aeruginosa is a leading cause of nosocomial and serious life-threatening infections and infections caused by this bacterium continue to pose a major medical challenge worldwide. The ability of P . aeruginosa to produce multiple virulence factors and in particular to form biofilms makes this bacterium resistant to all known antibiotics. As a consequence, standard antibiotic therapy are increasingly become ineffective to clear such infections associated with biofilms. In search for novel effective agents to combat P . aeruginosa biofilm infections, a series of the BmKn‒2 scorpion venom peptide and its truncated derivatives were synthesized and their antibiofilm activities assessed. Among the peptides tested, BmKn‒22 peptide, which was a modified peptide of the parental BmKn‒2 scorpion venom peptide, clearly demonstrated the most potential inhibitory activity against P . aeruginosa biofilms without affecting the bacterial growth. This peptide was not only capable of inhibiting the formation of P . aeruginosa biofilms, but also disrupting the established biofilms of P . aeruginosa . Additionally, BmKn‒22 peptide was able to inhibit the production of key virulence factor pyocyanin of P . aeruginosa . Our results also showed that BmKn‒22 peptide significantly reduced lasI and rhlR expression, and suggested that BmKn‒22 peptide-mediated inhibition of P . aeruginosa biofilms and virulence factors was achieved through the components of quorum-sensing systems. Combination of BmKn‒22 peptide with azithromycin resulted in a remarkable reduction P . aeruginosa biofilms. Since this peptide exhibited low toxicity to mammalian cells, all our results therefore indicate that the BmKn‒22 peptide is a promising antibiofilm agent against P . aeruginosa and warrant further development of this peptide as a novel therapeutic for treatment of P . aeruginosa ‒associated biofilm infections.