Pleurotus ostreatus is able to bioaccumulate several metals in its cell structures; however, there are no reports on its capacity to bioaccumulate iron. The objective of this study was to evaluate cultivation variables to increase iron bioaccumulation in P. ostreatus mycelium. A full factorial design and a central composite design were utilized to evaluate the effect of the following variables: nitrogen and carbon sources, pH and iron concentration in the solid culture medium to produce iron bioaccumulated in mycelial biomass. The maximum production of P. ostreatus mycelial biomass was obtained with yeast extract at 2.96 g of nitrogen L −1 and glucose at 28.45 g L −1 . The most important variable to bioaccumulation was the iron concentration in the cultivation medium. Iron concentration at 175 mg L −1 or higher in the culture medium strongly inhibits the mycelial growth. The highest iron concentration in the mycelium was 3500 mg kg −1 produced with iron addition of 300 mg L −1 . The highest iron bioaccumulation in the mycelium was obtained in culture medium with 150 mg L −1 of iron. Iron bioaccumulation in P. ostreatus mycelium is a potential alternative to produce non-animal food sources of iron.