Encapsulation of clove essential oil (CEO) by chitosan nanoparticles (ChNPs) was performed, using an emulsion-ionic gelation technique to improve the antifungal efficacy of CEO. The mass ratios of chitosan (Ch) to tripolyphosphate (TPP), 1:1, for unloaded ChNPs and 1:1:1 for Ch to TPP to CEO, for CEO-loaded ChNPs (CEO-ChNPs), were selected as optimum formulations based on dynamic light scattering and ultraviolet-visible spectroscopy. The presence of CEO in optimum CEO-ChNPs, was evidenced by Fourier transform infrared spectroscopy. Particle size distribution, of around 40 and 100 nm for the most optimum unloaded and oil-loaded ChNPs, was obtained by field emission-scanning electron microscopy. In vitro release studies of CEO-ChNPs revealed a controlled release during 56 days. The nano-encapsulated CEO demonstrated a superior performance against Aspergillus niger, isolated from spoiled pomegranate, compared with ChNPs and free oil. Therefore, this study revealed that CEO-ChNPs can be used as a promising natural fungicide in agriculture and food industry.