Several techniques were used to investigate the mechanism of antibacterial effect of catfish epidermal mucus extracts (CEME) against Pseudomonas aeruginosa. The results showed that the inhibitory effect was significant when the concentration exceeded 0.15% (mg/mL). The bacterial growth curve showed a decrease and scanning electron microscopy (SEM) showed changes in morphology. This indicated that the primary mechanism of CEME inhibitory action was to damage the bacterial cell membrane integrity. CEME also increased bacterial membrane permeability, which led to leakage of cellular small molecules and a proportion of macromolecules. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that CEME causes obvious changes to bacterial membrane proteins, which led to inhibition of bacterial protein synthesis and subsequent decline and loss of intracellular protein content. These results imply that CEME shows relatively good antibacterial effect on Pseudomonas aeruginosa.