Blue mold caused by Penicillium italicum is one of the most devastating postharvest diseases in Citrus fruit. This work aims to determine the antifungal potential of (E)-2-octenal, a component of plant essential oil, on the mycelial growth and disease incidence of a prochloraz-resistant strain of P. italicum isolated from an orchard in Luxi, Hunan Province, China. An in vitro assay revealed that the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of (E)-2-octenal were 0.25 and 0.50 mL L⁻¹, respectively. An in vivo assay showed that wax + 4 × MFC (E)-2-octenal treatment could reduce the blue mold incidence in Ponkan fruit inoculated with P. italicum without impairing fruit quality. Scanning electron microscopy analysis illustrated that the mycelia of P. italicum were anomalously twisted and shrunken upon treatment with (E)-2-octenal. The primary antifungal targets were attributed to the cell membrane instead of the cell wall based on propidium iodide and calcofluor white staining results. In addition, (E)-2-octenal induced massive accumulation of reactive oxygen species in hyphae in a dose-dependent manner, leading to oxidative stress and dysfunction of energy and respiration metabolism. At the same time, the activities of phosphofructokinase (PFK) and pyruvate kinase (PK) were decreased in (E)-2-octenal-treated samples. Taken together, our present study suggested that (E)-2-octenal could exert its antifungal activity against P. italicum by damaging the function of mitochondria and may be a potential antifungal substance for an alternative to chemical fungicides.