Abstarct: The medicinal mushroom Hericium erinaceus has attracted considerable attention from researchers due to its antioxidant properties and bioactive compounds. This study investigated the effect of magnesium oxide (MgO) nanoparticles on the production of phenolic compounds, antioxidant activity, and cytotoxicity.In the first phase, using a one-factor-at-a-time method, glucose, soybean powder, meat peptone, MgO nanoparticles, and a pH of 8 were identified as significant factors affecting the production of phenolic compounds. In this research, iron oxide (Fe3O4) nanoparticles were also examined, but due to their insignificant effects on the results, they were excluded from futher studies. Subsequently, experimental design using the statistical method RSM indicated that a culture medium containing 40 grams per liter of glucose, 0.14 grams per liter of MgO nanoparticles, and 15 grams per liter of meat peptone and soybean powder (in equal ratios) resulted in the highest total phenolic content production.The results of the antioxidant activity assessment showed that the fruiting body extract of the mushroom exhibited the highest DPPH radical scavenging activity with an IC50 value of 0.119 mg/mL. Identification of phenolic compounds in the mycelium and fruiting body revealed that the fruiting body had greater diversity, with 20 identified compounds (including protocatechuic, caffeic, and ferulic acids) compared to 17 compounds found in the mycelium.In the evaluation of erinacines, all types of erinacines were identified in the mycelium, while the fruiting body contained only 9 types of these compounds (A to G, I, J, and S). The cytotoxicity assessment indicated that the mycelium extract cultivated in a medium enriched with MgO nanoparticles exhibited the highest inhibitory effect, with an IC50 value of 155 µg/mL.