Abstract: The antifungal mechanism of trans-cinnamaldehyde (TC) against Villosiclava virens was investigated. The alteration of cell ultrastructure was observed using transmission electron microscope. The integrity of cell wall and activities of nicotinamide adenine dinucleotide-malic dehydrogenase (NAD-MDHase), succinate dehydrogenase (SDHase) and adenosine triphosphatase (ATPase) of V. virens were determined after treated with TC at the mass concentration of 2, 4, 8, 15 and 30 μg/mL. The results showed that the contents of lipidosome increased significantly and the structure of mitochondrion was indiscernible after treatment with TC at the mass concentration of 4 μg/mL. With the mass concentration of TC increasing to 30 μg/mL, the integrity of cell wall in V. virens was destroyed and the relative inhibition of NAD-MDHase and SDHase were reduced by 44.3% and 76.7%, respectively. Differently, the activities of Na⁺-K⁺-ATPase, Ca²⁺-Mg²⁺-ATPase, Ca²⁺-ATPase and Mg²⁺-ATPase fluctuated as U-curve with the concentration increase of TC. When the mass concentration of TC was 4 μg/mL, the highest inhibitive activity of TC on ATPase was the achieved among all the tested concentrations and the relative inhibition of TC on ATPase was higher than 78.4%. These results suggested that the cell wall and mitochondrion were the possible action targets for TC to inhibit the mycelial growth.