The fruiting bodies of Ganoderma sinensis are used as food raw materials of marketed healthcare products. To gain an insight into the chemical and biological profling of G. sinensis, twenty-five compounds including eleven undescribed ones (ganodermasines A‒K) and fourteen known ones were isolated. Among them, ganodermasines A‒D are undescribed diketopiperazine alkaloids, ganodermasines E and F are alkaloids containing a pyridine. The structures of undescribed compounds were identified by spectroscopic, computational, and crystallographic methods. The results of acetylcholinesterase (AchE) inhibitory activity show that ganodermasines I and J could inhibit AchE with IC50 values of 26.05 and 20.40 μM, respectively. In addition, neurotrophic assay in PC-12 cells showed that (+)-ganodermasine E, (−)-ganodermasine E, and ganodermasine I could stimulate neurite outgrowth at 10 μM, while the other isolates are inactive. The present findings will lend a hand for further utilization of G. sinensis. © 2022 Elsevier Ltd