Background: The genus Ganoderma includes about 80 species of mushrooms. Ganoderma lucidum is the best-known fungal species in mycotherapy and likely has the highest number of studies. Numerous bioactive compounds seem to be responsible for its beneficial effects; in particular, triterpenes, peptidoglycans and polysaccharides are the main physiologically active constituents. The aim of the present work is to identify the main bioactive components in aqueous extracts of G. lucidum spores obtained by two different extraction processes. Methods: The spores were at first extracted by a Soxhlet apparatus with n-hexane and the aqueous solutions were submitted to two different procedures: hot water extraction (HWE) and rapid solid-liquid dynamic extraction (RSLDE) using the Naviglio extractor. The extracts were then dialyzed to separate the compounds with higher molecular weight from polar compounds with lower molecular weight. The aqueous extracts and dialyzed fractions were tested on two human cell lines: human colonic epithelial cells (Caco-2) and human colorectal carcinoma cells (HTC-116). Results: GC-MS and NMR data revealed the presence of a mixture of glucose and mannitol in ratio 3.7:1 in the most active fraction. Conclusions: The outside dialysis phase of RSLDE extract seems to be particularly cytotoxic for HTC-116 and, interestingly, solutions with different concentrations of pure mannitol showed cytotoxic activity against this cell line too, although to a lesser extent.