Lipid droplets (LDs), cytosolic fat storage organelles, are emerging as major regulators of lipid metabolism, trafficking, and signaling in various cells and tissues. LDs are altered in cardiovascular and neuronal disorders, inflammation, obesity, and cancer. Flavonoids comprise different classes of molecules, characterized by a well-known antioxidant activity and a beneficial effect in several diseases. However, the cellular mechanism by which different classes of flavonoids improve health is poorly understood, in particular as far as LDs biogenesis is concerned. Here we used Drosophila melanogaster as a model system to investigate the effects of a selected group of flavonoids on larval tissues by examining LDs biogenesis. In our study, fruit flies were grown in xanthohumol-, isoquercetin-, and genistein-enriched food and larval tissues were analyzed using a LD marker. Total mRNA expression of two main enzymes (minotaur and midway) responsible for triacylglycerides synthesis was evaluated after treatments. Among the flavonoids analyzed, xanthohumol and isoquercetin resulted to be potent regulators of LDs biogenesis in a tissue-specific manner, inducing fat storage decrease in fat bodies and accumulation of LDs in nerves. Since LDs have been suggested to play a protective role against intracellular stress in nonadipocyte cells, our data support the hypothesis that some phytochemicals could act as strong modulators of LDs biogenesis in vivo. The knowledge of how different flavonoids act on lipid metabolism in different tissues can help to manage the use of phytochemicals with the aim of selectively ameliorating specific neuronal and metabolic diseases’ manifestations.