Iminosugars have a carbohydrate-like backbone in which the ring oxygen is replaced by nitrogen. They are naturally found in foods such as rice, buckwheat, mulberries, and fermented vegetables, and are reported to exert anti-hyperlipidemic and anti-hyperglycemic effects due to the inhibition of cellular glycosidases. This mechanism suggests their potential role in cancer treatment and prevention. In this study, two natural iminosugars, D-fagomine (FGM) and 1-deoxynojirimycin (DNJ), and their synthetic derivatives were screened for potential anticancer properties using Caco-2 and HCT-116 cells as models for the early and late stages of colon cancer, respectively. Iminosugars were found to decrease cell viability, with effects varying based on the type of iminosugar, cell type, growth condition (glucose concentration), exposure time (1 vs. 13 days), and tissue architecture (monolayer vs. spheroid). The combined use of innovative techniques, such as IncuCyte^® live cell imaging and Seahorse real-time cellular metabolic analysis, and microscopic observation after staining enabled us to detect changes in substrate utilization for energy metabolism, including increased glycolysis and alterations in lipid and glycogen stores. The evidence that iminosugars, both natural and synthetic, influence cellular bioenergetics paves the way for their potential use in various applications, including cancer treatment.