Diatoms are microscopic biorefineries producing value-added molecules, including unique pigments, triglycerides (TAGs) and long-chain polyunsaturated fatty acids (LC-PUFAs), with potential implications in aquaculture feeding and the food or biofuel industries. These molecules are utilized in vivo for energy harvesting from sunlight to drive photosynthesis and as photosynthetic storage products, respectively. In the present paper, we evaluate the effect of narrow-band spectral illumination on carotenoid, LC-PUFAs and TAG contents in the model diatom Phaeodactylum tricornutum. Shorter wavelengths in the blue spectral range resulted in higher production of total fatty acids, namely saturated TAGs. Longer wavelengths in the red spectral range increased the cell’s content in Hexadecatrienoic acid (HTA) and Eicosapentaenoic acid (EPA). Red wavelengths induced higher production of photoprotective carotenoids, namely fucoxanthin. In combination, the results demonstrate how diatom value-added molecule production can be modulated by spectral light control during the growth. How diatoms could use such mechanisms to regulate efficient light absorption and cell buoyancy in the open ocean is discussed.