The work investigated the effect of Lithovit-Urea50 on the composition of Pleurotus ostreatus (Jacq. Ex Fr.) P. Kumm. (1871) cultivated on spent oyster substrate mixed with wheat straw (1:1, w/w mixture). The product was applied in different doses (C1: 3 g kg-1 and C2: 5 g kg-1) at three distinct timings (t1: at spawning, t2: after first harvest, t3: at spawning and after first harvest). Protein and fiber contents increased respectively by 0.64 and 0.2% in C1t1 and by 0.46 and 0.8% in C2t2 compared to control (C0t0). Total carbohydrates increased by 0.48-3.76%. Sucrose and glucose contents decreased in the majority of treatments, while fructose increased in C2t1 (by 0.045%). Essential amino acids were the highest in C1t1, wherein respective improvement of 0.31, 0.10, 0.05, 0.21, 0.18, and 0.09% compared to C0t0. Similarly, C1t1 was superior in non-essential amino acids. Potassium, sodium, calcium, iron, and copper contents decreased in all treatments, with minor exceptions, zinc decreased in C1t1 and C2t1, while nickel and lead increased in all treatments. Conclusively, despite important ameliorations in the mushroom nutritional value, mostly in C1t1, the product should be further tested in lower doses (< 3 g kg-1) to counteract its effect on heavy metal bioaccumulation.