This work aimed to investigate the antibacterial ability and potential mechanism of chitosan grafted gentisate acid derivatives (CS-g-GA) against Pseudomonas fluorescens. The results showed that CS-g-GA had a significant suppressive impact on the growth of Pseudomonas fluorescens, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 0.64 mg/mL and 1.28 mg/mL, respectively. Results of scanning electron microscopy (SEM) and alkaline phosphatase (AKPase) confirmed that CS-g-GA destroyed the cell structure thereby causing the leakage of intracellular components. In addition, 1 × MIC of CS-g-GA could significantly inhibit the formation of biofilms, and 74.78 % mature biofilm and 86.21 % extracellular polysaccharide of Pseudomonas fluorescens were eradicated by CS-g-GA at 2 × MIC. The results on the respiratory energy metabolism system and antioxidant system demonstrated that CS-g-GA caused respiratory disturbance and energy limitation by influencing the key enzyme activities. It could also bind to DNA and affect genetic metabolism. From this, it could be seen that CS-g-GA had the potential to control foodborne contamination of Pseudomonas fluorescens by attacking multiple targets.