The aim of this study was to evaluate the effect of high selenium (Se) concentrations on morphophysiological and ultrastructural properties ofPleurotus ostreatus. Mycelium growth was good in media enriched with 5.0, 10.0, and 20.0 mg L−1of Se, concentration of 500.0 mg L−1strongly inhibited growth, and 1000.0 mg L−1was the minimum inhibitory concentration. Contrary to thin-walled, hyaline, branched, and anastomized hyphae with clamp-connections in the control, at Se concentrations of 100.0 and 500.0 mg L−1, they were noticeably short, frequently septed and branched, with a more intensive extracellular matrix, and without clamp-connections. At high Se concentrations, hyphae with intact membrane, without cellular contents, with a high level of vacuolization, and with numerous proteinaceous bodies were observed. Biomass yield ranged between 11.8 g L−1, in the control, and 6.8 g L−1, at an Se concentration of 100.0 mg L−1, while no production was detected at a concentration of 500.0 mg L−1. Se content in the mycelia reached a peak (938.9 μg g−1) after cultivation in the medium enriched with Se at the concentration of 20.0 mg L−1, while the highest absorption level (53.25%) was found in the medium enriched with 5.0 mg L−1Se.