Abstract: To investigate the chemical composition of tea tree essential oil（TTO）and its antibacterial activity against pathogens causing skin ulceration syndrome in sea cucumber（Apostichopus japonicus）, the chemical constituents of several TTO batches were analyzed and relatively quantified using gas chromatography–mass spectrometry（GC–MS）coupled with the peak area normalization method. A batch with high content of active components was selected for antibacterial tests against the pathogens. The results indicated that the TTO from Beihai, Guangxi（Batch No. 20230503）exhibited the highest total content of active components（89.17%）, including terpinen-4-ol（38.44%）, γ-terpinene（21.32%）, 1,8-cineole（1.31%）, α-terpinene（11.63%）, and α-pinene（3.01%）. Consequently, this batch was chosen for the subsequent antibacterial study. In the disk diffusion assay, the inhibition zone diameters against Vibrio harveyi, Vibrio alginolyticus, and Pseudoalteromonas sp. were 36.50 ± 3.2 mm, 26.36 ± 1.2 mm, and 31.85 ± 4.0 mm, respectively. The minimum inhibitory concentrations（MICs）of the TTO-based biogenic inhibitor against these pathogens were 30, 40, and 30 mg·mL⁻¹, respectively, while the minimum bactericidal concentrations（MBCs）were 20, 20, and 15 mg·mL⁻¹, respectively. In the safety assessment on A. japonicus, TTO concentrations below 100 mg·mL⁻¹ showed no adverse effects on sea cucumber growth, and no chemical residues were detected, demonstrating its safety profile and potent antibacterial efficacy.