Iron biofortification of Hericium erinaceus is a promising strategy for enhancing its nutritional profile. This study examined the effects of supplementing growth substrates with various iron forms (FeCl3⋅6H2O, FeSO4⋅7H2O, and FeHBED) on iron uptake, accumulation, and metabolic profiles in H. erinaceus fruiting bodies. The results indicated that iron supplementation not only increased iron content but also affected several metabolic path- ways, including energy metabolism, oxidative stress response, and biosynthesis. The choice of iron significantly influenced mushroom growth, mineral composition, and metabolite levels. FeCl3⋅6H2O resulted in the highest iron accumulation, FeSO4⋅7H2O enhanced biomass production, and FeHBED (water-soluble iron chelate) exhibited unique effects on energy metabolism and antioxidant protection. These findings suggest that iron biofortification could facilitate the development of nutritionally enhanced H. erinaceus products, potentially addressing iron deficiency while preserving the medicinal properties of mushrooms. Further research is necessary to optimise iron biofortification techniques and assess the bioavailability and health benefits of iron-enriched H. erinaceus in human nutrition.