The antifungal activity of a natural resorcylic acid lactone, hypothemycin (HPM), against Peronophythora litchii in vitro and in vivo was investigated. HPM treatment substantially suppressed spore germination of P. litchi, with the inhibition rate of 100% when 0.78 μg/mL HPM was applied. Similarly, mycelial growth of P. litchii was efficiently inhibited. Furthermore, HPM caused the ultrastructural modifications of P. litchii, including the disruption of the cell wall and the endomembrane system, especially the plasma membrane, mitochondria, and vacuoles, which led to the destruction of the cellular integrity. Moreover, application of HPM significantly reduced decay and suppressed peel browning of postharvest litchi fruit inoculated with P. litchii during storage at 28 °C. Overall, these findings suggested that HPM exhibited excellent antifungal activity against P. litchii both in vitro and in vivo, which could be helpful for the storage of harvest litchi fruit.