The development of natural essential oil as an alternative to synthetic chemicals in the control of postharvest decay is currently in the spotlight. In the present study, the efficacy of seven essential oils in suppressing Collelotrichum gloeosporioides identified from sweet cherry was evaluated in vitro and clove oil was proved to be the most promising inhibitor. Thus, the antifungal properties and potential mechanisms of clove oil in vitro and in vivo by fumigation and contact treatments were intensively investigated. For C. gloeosporioides, the minimal inhibitory concentrations (MIC) of clove oil in air and contact phase were 80 and 300 μL/L in vitro testing, respectively. Based on the radial growth of C. gloeosporioides mycelium in medium, the fumgitoxic ability of essential oil was observed in a dose-dependent manner, which was not as dramatic as that under in vivo conditions. Furthermore, scanning electron microscopy and transmission electron microscopy of C. gloeosporioides exposed to clove oil exhibited obviously deleterious morphological and ultrastructural alterations confirming the disruption of fungal cell wall and endomembrane system, which resulted in increasing in permeability and causing the loss of intracellular constituents. In future, essential oils, combined with nano-emulsification approaches, could be good candidates as safe and effective antifungal agents for fungal spoilage of fresh commodities.