Used in the synthesis of vitamin D2, ergostenol plays a key role in pharmaceutical and nutritional industries. When exposed to ultraviolet light, it undergoes photochemical conversion to produce ergocalciferol (vitamin D2), which is used to fortify foods and treat vitamin D deficiency. It also serves as a biomarker in environmental and microbiological studies to estimate fungal biomass in samples such as soil, air, and indoor environments. Due to its presence in fungal cell membranes, it helps researchers assess mold contamination and indoor air quality. Additionally, ergostenol is used in research related to membrane biology and sterol function in eukaryotic cells.