Human serum paraoxonase 1 (hPON1) which hydrolyzes Pseudomonas aeruginosa acyl homoserine lactone (AHL) signal molecules was used as antibiofilm agent. hPON1 was purified by using ammonium sulfate precipitation and specially designed hydrophobic interaction chromatography (Sepharose 4B-L-tyrosine-1-Naphthylamine) from the fresh human serum. As cell motility of swarming, swimming and twitching are proven instrumental in biofilm formation, we investigated whether or not hPON1 affected the P. aeruginosa motility. hPON1 was reduced the early stage of biofilm formation, mature biofilm and motilities. The early stage and old biofilm were decreased more than 50% by 1 mg ml(-1) of hPON1 concentration within range of 0.1-10 mg ml(-1). Additionally, exopolymeric substance (EPS) of mature biofilm was indirectly decreased by hPON1. Inhibitory effect of hPON1 within range of 0.003-30 mg ml(-1) on swarming and swimming motilities. But it resulted in highly inhibitory effects on twitching motility at concentration as low as 0.3 mg ml(-1) concentration. This study proved that hPON1 alone can be safely used to inhibit/disrupt the mature biofilms and cell motility of P. aeruginosa and beholds much promise in clinical applications.