The antibacterial mechanism of chitosan-grafted caffeic acid (CS-g-CA) against Pseudomonas fluorescens (P. fluorescens) was studied. The results indicated that the MIC of CS-g-CA to P. fluorescens was 1.25 mg/mL, and CS-g-CA in MIC could effectively inhibit the growth of bacteria. CS-g-CA can greatly destroy the cell membrane of P. fluorescens, enhancing its permeability and leading to the leakage of macromolecular proteins. The results of reactive oxygen species (ROS) and lipid peroxidation activities demonstrated that CS-g-CA could destroy the respiratory and metabolic system. According to scanning electron microscope (SEM), CS-g-CA can destroy the somatic cells of bacteria, resulting in the leakage of their contents and death by lysis. In addition, thallus movement was inhibited by CS-g-CA. Electrophoresis results showed that the DNA of P. fluorescens with CS-g-CA treatment was destroyed, which further led to the death of microorganisms. Eventually, the normal growth and reproduction of bacteria were also hindered. These findings suggested that CS-g-CA is a promising antimicrobial agent to control P. fluorescens in aquatic products.