This study aimed to examine the inhibition of chlorogenic acid-grafted chitosan (CS-g-CA) on Pseudomonas fluorescens (P. fluorescens) and its biofilm. The minimum inhibitory concentration (MIC) of CS-g-CA against P. fluorescens was 1.25 mg/mL. Alkaline phosphatase (AKPase) leakage assay and scanning electron microscopy (SEM) observation showed that CS-g-CA causes structural damage to cell walls and membranes, resulting in the loss of function. In addition, CS-g-CA was able to disrupt the antioxidant system of P. fluorescens, interfere with energy metabolism, and interact with genomic DNA, affecting the normal physiological function of bacteria. It was also found that CS-g-CA inhibited the flagellar motility of P. fluorescens, which may be responsible for the inhibition of its biofilm formation. CS-g-CA at 2MIC was able to remove 71.64% of the mature biofilm and reduce the production of extracellular polysaccharides (EPS) by 60.72%. This was further confirmed by confocal laser scanning microscopy (CLSM), which showed a significant reduction in the amount of biofilm. In summary, CS-g-CA has strong antibacterial and anti-biofilm activities against P. fluorescens, and it can be applied as a potential seafood bacteriostatic agent.