In this article, in vitro and in vivo antifungal activities of clove oil to inactivate Aspergillus flavus, Penicillium citrinum and Rhizopus nigricans were investigated. The result showed that eugenol (approximately 83.02%) is the main constituent of clove oil. The minimum inhibitory concentrations of clove oil against A. flavus, P. citrinum and R. nigricans were 25, 25 and 50 mL/mL, respectively. In an in vitro experiment, the results revealed that clove oil possessed stronger antifungal activities against A. flavus and P. citrinum than against R. nigricans, and the antifungal activity improved with increasing its concentration. In an in vivo study, clove oil treatments significantly reduced fungal decay, and clove oil at a concentration of 3.0% showed complete control of A. flavus and P. citrinum in wound-inoculated fruit. These results indicated that clove oil has a good potential to be a natural antifungal agent for fruit applications. PRACTICAL APPLICATIONSPostharvest decay caused by fungi is the major cause of rapid deterioration of fruits, which affects the quality and shortens the shelf life of fruits and vegetables. The major problems for the application of chemical preservatives are the increasing resistance to antibiotics and chemotherapeutics and health risk concerns. So, most consumers demand less use of synthetic additives to ensure food safety. There is a growing need to develop alternative approaches, such as the application of essential oils, for controlling postharvest diseases.