Plant-derived essential oils have excellent antifungal effects and can be used for the preservation of fresh foods such as fruits and vegetables, but the detailed mechanism has not been fully elucidated. In this study, we investigated the inhibitory effects of trans-cinnamaldehyde on Penicillium italicum, a common pollution fungus in citrus, and explored the antifungal mechanism of trans-cinnamaldehyde by detecting fungal oxidative damage, mitochondrial metabolism, and cell apoptosis. These results showed that trans-cinnamaldehyde made the carboxylic acid cycle deregulated by altering the related enzyme activities (succinate dehydrogenase, malate dehydrogenase) and mid product. Moreover, the level of reactive oxygen species rose sharply while the redox level was out of regulation. The mitochondrial membrane potential collapsed, leading to the leakage of cytochrome c, and then triggering the activation of apoptotic protease, which was further confirmed by the significant increase in caspase-3 activity from (3.6 ± 0.6) U to (8.8 ± 1.1) U (P < 0.05). The cytochrome c in mitochondria was detected by confocal Raman microspectroscopy, the characteristic intensity index (I750/I2944) was decreased, indicating that the cytochrome c in mitochondria was reduced and leakage. Besides, the strong negative correlation between Raman intensity and the amount of cytochrome c leakage was established with the correlation coefficient of -0.9817. This study revealed that destroying the integrity of the mitochondrial membrane, activating the mitochondrial-mediated apoptosis pathway was the in-depth antifungal mechanism of trans-cinnamaldehyde; and Raman spectroscopy technology provided new ideas to study this process with high sensitivity determination of cytochrome c.