Abstract: To explore the characteristic functional components and the potentiality of high-value utilization, five different Castanea mollissima cultivars (‘Xinzaoli’, ‘Zaofeng’, ‘Dahongpao’, ‘Qinli No.2’, and ‘Taishan No.1’) were selected to investigate their functional active components and in vitro enzyme inhibitory activities in different fruit parts (kernel, seed coat, shell, and bur). The results showed that the contents of total phenol (7.15~14.59 mg GAE/g DW) and total flavonoid (8.65~20.06 mg RUE/g DW) in seed coats were significantly higher than these of other fruit parts in all C. mollissima cultivars. And the highest contents of total phenol and total flavonoid were both found in the seed coat extract of ‘Xinzaoli’. According to the quantitative analysis of the main functional components by LC-MS, rutin (0.14~130.05 mg/100 g DW), ellagic acid (8.53~823.44 µg/100 g DW), p-hydroxybenzoic acid (9.03~73.95 µg/100 g DW), and gallic acid (7.97~231.55 µg/100 g DW) were the major active constituents in all detected C. mollissima samples. Except for cholesterol esterase inhibitory activity, the extracts of seed coat from all C. mollissima cultivars exhibited the strongest enzyme inhibitory activities, followed by bur, shell, and kernel extracts. Notably, the extracts of seed coat within ‘Xinzaoli’ showed the highest enzyme inhibitory activities in α-glucosidase (262.81 µg ACE/g DW), tyrosinase (6.42 mg KAE/g DW), and xanthine oxidase (2.03 mg ALE/g DW). Differently, the extracts of seed coat in ‘Taishan No.1’ displayed the strongest acetylcholinesterase inhibitory activity (1535.99 µg DHE/g DW). For cholesterol esterase, the highest inhibitory activity was observed in the extracts of bur (6.63~8.09 ng ORE/g DW), followed by seed coat (3.66~5.06 ng ORE/g DW), shell (1.00~2.51 ng ORE/g DW), and kernel extracts (0.82~0.97 ng ORE/g DW). Among the C. mollissima cultivars, the extracts of bur within ‘Zaofeng’ showed the highest inhibitory effect on cholesterol esterase (P<0.05). Further, the results of multivariate statistical analysis (PLS-DA and correlation analysis) indicated that rutin, total phenols, total flavonoids, and tyrosinase inhibitory activity were key biomarkers in distinguishing the different part samples. Epicatechin, catechin, quercetin, rutin, p-hydroxybenzoic acid, and gallic acid were significantly correlated with the enzyme inhibitory activities of the by-products of chestnut processing (seed coat, shell, and bur), which can be considered as the key functional components. This study clarifies the main functional active components and the characteristics of five enzyme inhibitory activities in each fruit part of different C. mollissima cultivars. Furthermore, the seed coat and bur extracts had the relatively strong biological activities. These results would provide a scientific basis for the development of functional products in chestnut, and the resource utilization of the by-products of chestnut processing.