Previously, we had prepared acetyl phenyl-thiosemicarbazone derivatives of chitosan, and their antimicrobial activities were analyzed. The purpose of the present study was to further assess the relationship between the structure and antimicrobial activities of benzoyl phenyl-thiosemicarbazone-chitosan. Ten new benzoyl phenyl-thiosemicarbazone-chitosans were prepared and their structures were characterized by FT-IR and elemental analysis. The antimicrobial experiment against four species of bacteria and four crop-threatening pathogenic fungi were conducted based on the derivatives of chitosan with different molecular weight at different concentrations. The results indicated that the antimicrobial activities of benzoyl phenyl-thiosemicarbazone derivatives are much better than that of pure CS. The value of the minimum inhibition concentration (MIC) and the minimum bactericidal concentration (MBC) of the derivatives against Escherichia coli was 7.03 and 225 μg mL(-1) respectively. All of the derivatives had significant inhibiting effect on the investigated fungi in the concentration of 50-500 μg mL(-1), and the maximum inhibitory index was 94.74%. These results indicate that the derivatives have potential ability used as antibacterial reagent in agricultural field.