There are kinds of food preservatives applied in the food industry， however， most of them only aim at gram-positive bacteria and have no effects on gram-negative bacteria. In order to develop new food preservatives with good antibacterial activity， molecular docking was carried out using UCSF DOCK6.5. The penicillin-binding protein 3 （PBP3） of Pseudomonas aeruginosa and PBP1b， PBP4 and PBP5 of Escherichia coli were chosen as the targets. ZINC database， containing 5 800 000 drug-like small molecules， was used for the virtual screening. After analysis， one of the hits was selected as lead compound and subjected to organic synthesis and food preservation. Compared with potassium sorbate， the lead compound was more effective on the control of pH value， total number of bacterial colonies， drop loss and TVB-N of chilled pork except color protection. But both of them had better results than control group. The research results proved that the lead compound had a better preservation effect on chilled pork， and it was expected to further develop it into a new food preservative.