The effects of selenium enrichment on the biological efficiency, phenolic compounds, amino acid profile, antioxidant capacity, and cellular antioxidant activity (CAA) were evaluated in Pleurotus ostreatus fruiting bodies (FB) harvested during three sequential flushes. Sodium selenate was used to reach selenium content of 17.5 or 5.8 mg/kg in the sorghum straw substrate. Biological efficiency and total selenium content increased. One of the main differences among treatments was in ergothioneine content, an indicator of oxidative stress that was positively related with valine and isoleucine contents and negatively related to leucine and phenylalanine. Besides ergothioneine, nucleosides derived from adenine and uracyl were the major peaks observed in all treatments, and coumaric and ferulic acids were found in the bound phenolics extract. Selenium enrichment also affected the antioxidant capacity, and particularly the methanolic extract obtained from the second flush of FB cultivated in selenium-enriched substrate (17.5 mg/kg) had the best CAA.