The species Pleurotus ostreatus is a commercially, gastronomically, and biotechnologically important fungus. Its strain variability has been little researched. The study provides an evaluation of 59 oyster mushroom production strains in terms of the ability to accumulate selected metals in the cap and stipe. The fruiting bodies were grown under identical model conditions on straw substrate. Metal concentrations (ET-AAS) in dry fruiting bodies ranged in values 1.7–22.4 mg kg−1 for Al, 2.6–9.7 mg kg−1 Ba, 199–4560 mg kg−1 Ca, 1.7–12.0 mg kg−1 Cu, 12–120 mg kg−1 Fe, 16,000–49,500 mg kg−1 K, 876–2400 mg kg−1 Mg, 0.39–11.0 mg kg−1 Mn, 46–920 mg kg−1 Na and 11–920 mg kg−1 for Zn. More Cu, Fe, K, Mg, Mn, Zn accumulated in the cap, while in the stipe Ba was amassed. No significant difference was found between Al, Ca and Na between the accumulation in the cap and the stipe. Furthermore, the dependence of metal uptake from the substrate depending on the fortification of the substrate was confirmed. Statistically significant (p < 0.05) synergistic relationships were shown in pairs Al and Ba, Al and Fe, Ba and Na, Ba and Ca, Ca and Na, Cu and Fe, Fe and Mn, Fe and Zn, K and Mg, K and Mn, K and Zn, Mg and Mn, Mg and Na, Mg and Zn and Mn and Zn in the substrate without the addition of sodium selenate to the substrate. Altered relationships were observed after the application of sodium selenate to the substrate, synergism of Se and Ni, Se and Co and Se and Hg, Cu and Mn, Cu and Fe, Zn and Co, Zn and Ni, Zn and Hg, Mn and Fe, Mn and Cr, Co and Ni, Co and Hg, Ni and Hg, Pb and Cd. The findings of the study may help in the selection of production strains with hypercumulative properties for a particular metal and subsequent use in the addition of fortified fruiting bodies (e.g., with Zn). Based on the study the strains less sensitive to the accumulation of hazardous metals is possible to select for large-scale production, which is important from the perspective of food safety.