Biofilm formation is an important reason for bacterial resistance to antimicrobials. Many compounds with dihydro-pyrrol-2-one (DPO) have antibacterial effects. It is prospective to base on DPO skeleton to design new compounds for biofilm inhibition. DPO was designed by a novel method of tandem cyclization between ethyl glyoxalate and amines, the series of DPO derivatives were synthesized by change of the amines. Their activities were evaluated by the inhibition of biofilm in Pseudomonas aeruginosa. The interaction of DPO derivatives with mannitol dehydrogenase (MDH) or extracellular DNA (eDNA) in the biofilm was simulated by molecular docking to reveal possible mechanism. 19 new DPO derivatives were synthesized and identified, 15 of them had antibacterial activities, but only 5 of them had more than 50% inhibition on biofilm of P. aeruginosa at 50μg/mL. The MDH activity and eDNA content in biofilm decreased significantly after treatment of the DPO derivatives in concentration dependence. The simulation reveals that strong interaction exists between the five DPO derivatives and MDH or eDNA, which are involved in anti-biofilm mechanism. The synthetic method of DPO derivatives is practical to provide effective anti-biofilm agents for P. aeruginosa, and they take effect through inhibition on MDH and eDNA of biofilm.