The antifungal mechanism of essential oils against fungi remains in the shallow study. In this paper, antifungal mechanism of trans-cinnamaldehyde against Penicillium italicum was explored. Trans-cinnamaldehyde exhibited strong mycelial growth inhibition against Penicillium italicum, with minimum inhibitory concentration of 0.313 μg/mL. Conventional analytical tests showed that trans-cinnamaldehyde changed the cell membrane permeability, which led to the leakage of some materials. Meanwhile, the membrane integrity and cell wall integrity also changed. Surface-enhanced Raman spectroscopy, an ultrasensitive and fingerprint method, was served as a bran-new method to study the antifungal mechanism. Characteristic peaks of supernatant obviously changed at 734, 1244, 1330, 1338 and 1466 cm⁻¹. The Raman intensity represented a strong correlation with results from conventional methods, which made SERS an alternative to study antifungal process. All evidences implied that trans-cinnamaldehyde exerts its antifungal capacity against Penicillium italicum via membrane damage mechanism.