The antibacterial activity of terpinene-4-ol against Pseudomonas fluorescens was investigated by measuring the minimum inhibitory concentration (MIC) and growth curve. Its effect on cell wall and cell membrane permeability were evaluated by determination of alkaline phosphatase activity and electrical conductivity, fluorescein diacetate (FDA) staining and scanning electron microscope (SEM) observation, and its effect on intracellular macromolecules and cellular metabolism was further explored by determining DNA, total protein and Na⁺, K⁺-ATPase activity. The results showed that the MIC was determined to be 2 μL/mL. Terpinen-4-ol could effectively destroy the cell wall of P. fluorescens in a dose-dependentmanner, and enhance cell membrane permeability, leading to the leakage of intracellular ions. The results of SEM showed that terpinen-4-ol could damage the cell membrane irreversibly. Gel retardation electrophoresis analysis revealed that terpinene-4-ol could cause leakage of macromolecular DNA. Additionally, terpinen-4-ol reduced intracellular protein content and blocked the expression of protein and the synthesis of ATPase, thus resulting in cell apoptosis. Therefore, terpinen-4-ol has the potential to be used as a new preservative for aquatic products.