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In this study, antagonistic endophytic fungi were isolated from postharvest chestnut fruits; endophytic antagonistic fungi and their combination of inhibitory effects on the fungal pathogen <i>Neofusicoccum parvum</i> were evaluated. A total of 612 endophytic fungi were isolated from 300 healthy chestnut kernels, and 6 strains out of them including NS-3, NS-11, NS-38, NS-43, NS-56, and NS-58 were confirmed as antagonistic endophytic fungi against <i>Neofusicoccum parvum</i>; these were separately identified as <i>Penicillium chermesinum</i>, <i>Penicillium italicum</i>, <i>Penicillium decaturense</i>, <i>Penicillium oxalicum, Talarmyces siamensis</i>, and <i>Penicillium guanacastense</i>. Some mixed antagonistic endophytic fungi, such as NS-3-38, NS-11-38, NS-43-56, and NS-56-58-38, exhibited a much stronger antifungal activity against <i>N. parvum</i> than that applied individually. Among them, the mixture of NS-3-38 showed the highest antifungal activity, and the inhibition rate was up to 86.67%. The fermentation broth of NS-3, NS-38, and their combinations exhibited an obvious antifungal activity against <i>N. parvum</i>, and the ethyl acetate phase extract of NS-3-38 had the strongest antifungal activity, for which the inhibitory rate was up to 90.19%. The NS-3-38 fermentation broth combined with a chitosan coating significantly reduced <i>N. parvum</i> incidence in chestnuts from 100% to 19%. Furthermore, the fruit decay and weight loss of chestnuts during storage were significantly decreased by the NS-3-38 fermentation broth mixture along with a chitosan coating. Therefore, a mixture of <i>P. chermesinum</i> and <i>P. decaturense</i> could be used as a potential complex biocontrol agent to control postharvest fruit decay in chestnuts.