Little is known on the antioxidant activity modulation in microalgae, even less in diatoms. Antioxidant molecule concentrations and their modulation in microalgae has received little attention and the interconnection between light, photosynthesis, photoprotection, and antioxidant network in microalgae is still unclear. To fill this gap, we selected light as external forcing to drive physiological regulation and acclimation in the costal diatom Skeletonema marinoi. We investigated the role of light regime on the concentration of ascorbic acid, phenolic compounds and among them flavonoids and their connection with photoprotective mechanisms. We compared three high light conditions, differing in either light intensity or wave distribution, with two low light conditions, differing in photoperiod, and a prolonged darkness. The change in light distribution, from sinusoidal to square wave distribution was also investigated. Results revealed a strong link between photoprotection, mainly relied on xanthophyll cycle operation, and the antioxidant molecules and activity modulation. This study paves the way for further investigation on the antioxidant capacity of diatoms, which resulted to be strongly forced by light conditions, also in the view of their potential utilization in nutraceuticals or new functional cosmetic products.