The antifungal activity and possible mode of action of pinocembrin isolated from propolis against Penicillium italicum were investigated. Pinocembrin exhibited strong antifungal activity against P. italicum in a dose-dependent manner. Respiration rates of P. italicum during spore germination and mycelial growth were significantly inhibited when exposure to pinocembrin. The respirations of mitochondria in state 2 and state 3 from mycelia were significantly inhibited in the presence of this compound. The phosphorylated adenosine nucleotides levels in hyphae of P. italicum treated with pinocembrin were significantly low and energy charge value became unstable. Unltrastructure of hyphae was seriously damaged with pinocembrin incubation for 24 h, which was further confirmed by the increase of relative ionic leakage and soluble protein loss of P. italicum mycelia treated with pinocembrin. It was concluded that pinocembrin inhibited the mycelial growth of P. italicum by interfering energy homeostasis and cell membrane damage of the pathogen. Pinocembrin would be a promising bioactive compound for treatment of P. italicum infections on postharvest citrus fruit.