The presented case study illustrates the possibility of adding value to the biological surplus remaining from the mushroom cultivation industry. In essence, the unused mushroom parts were submitted to UV-C irradiation, with the purpose of increasing the vitamin D2 content and validating its extraction. Vitamin D2 concentration in three different mushroom species (Agaricus bisporus, A. bisporus Portobello, and Pleurotus ostreatus) was obtained by high-performance liquid chromatography (HPLC), by means of an ultraviolet (UV) detector. The method was validated using an A. bisporus Portobello sample, and its reproducibility and accuracy were confirmed. Independently of the UV-C irradiation dose, the effect on vitamin D2 concentration was significant, allowing it to increase from less than 4 µg/g dry weight (dw) to more than 100 µg/g dw in all mushroom species. These results are good indicators of the feasibility of industrial surplus mushrooms as sustainable vitamin D2 food sources, besides contributing to strengthen the circularity principals associated to the mushroom production chain.