To explore the bactericidal effect of dimethyl dicarbonate(DMDC) on foodborne harmful microorganism， the bactericidal effect of DMDC on the planktonic and biofilm bacteria of Pseudomonas fluorescens and Pseudomonas lund of spoilage organism in fresh food were comparatively evaluated in the present study. Effects of DMDC on the bacterial activity， intracellular enzyme activity and mature biofilm of Pseudomonas were measured by using API ZYM kit， XTT kits and confocal microscope. Meanwhile， antibacterial activity of DMDC at different mass concentration in tomato and lettuce were performed. The results showed that DMDC had stronger bactericidal action against the two Pseudomonas compared with other three pathogens. The two Pseudomonas were inactivated by 250 mg/L DMDC treatment for 5 min， which was attributed to the decreasing activities of alanine araminase and naphthol-AS-BI-phosphohydrolas. In addition， mature biofilm of P. fluorescein and P. lund could be effectively destroyed. The population in biofilm cell decreased by 4.60 lg (CFU/cm2) and 4.15 lg(CFU/cm2)， respectively， and the cell activity significantly dropped， resulting in the increase of dead cell and the thinner biofilm structure， when treated by 250 mg/L DMDC. Moreover， the aerobic plant count (APC)， Pseudomonas number (PA)， and mold and yeast count (MYC) of tomato and lettuce were significantly reduced by treatment of DMDC at 250 mg/L. The APC and PA in tomato was reduced by 2.60 lg (CFU/cm2) and 1.73 lg (CFU/cm2)， and in lettuce was decreased by 2.56 lg(CFU/cm2)and 1.98 lg(CFU/cm2)， respectively， for 15 min treatment， accompanied by no detection of mold and yeast after 5 min treatment. Sensory qualities were not affected. Thus， DMDC significantly killed the planktonic and mature biofilm of two Pseudomonas， and has a good bactericidal effects when applied to the surface of two kinds of fruits and vegetables， which is expected to become a new cleaning agent in fruit and vegetable.