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Sweet chestnut fruits are popular fruits commercialized as fresh or processed ready-to-eat products. The major post-harvest problems associated with stored chestnut fruits are fungal rots, which cause major losses in fruit quality. The aims of this work were to determine the incidence, abundance and diversity of rots and fungi in three chestnut varieties (Longal, Judia and Martaínha) of Portugal, collected from an industrial plant, and to identify the stages of storage and processing where fungi and rots are more significant. Thirty-three chestnut samples from the three varieties were collected from different stages of industrial processing. Nuts were internally and externally inspected for damage, infestation and infection, and internal fungi were isolated and molecularly identified. The variety Martaínha was identified as the least susceptible to fungal growth, while Longal was the most susceptible. A high diversity of fungi was detected and identified. The dominant fungi were Mucor racemosus, Penicillium spp. (the causal agents of green rots), Ciboria batschiana (black rot) and Botrytis cinerea (gray rot). Gnomoniopsis smithogilvyi, the causal agent of brown rot, was also frequently detected. Sterilization with hydrothermal bath was effective in the elimination or reduction of most of the rot-causing fungi. These results could serve as a baseline for better monitoring fungal development and chestnut decay, and to develop effective management measures to control post-harvest chestnut rots.