The present study for the first time addressed whether the simultaneous presence of selenium, zinc and iron may have effects on the selenium uptake in the mycelia of the winter mushroom (also known as enoki), <i>Flammulina velutipes</i>. Response surface methodology was used to optimize concentrations of selenium, zinc and iron in the range of 0 to 120 mg L<sup>-1</sup>. The findings showed that application of selenium, zinc and iron (singly, in pairs, or triads) significantly enhanced the selenium accumulation in the mycelia. The highest amount of the selenium accumulation was observed when selenium (60 mg L<sup>-1</sup>) and zinc (120 mg L<sup>-1</sup>) were applied into submerged culture media, concurrently, leading to an 85-fold and 88-fold increase in the selenium content of the mycelia compared to that of the mycelia treated with selenium only and untreated mycelia, respectively. In addition, accumulation of selenium into the mycelia had no deteriorative effects on the mycelial biomass. The findings presented in this study may have implications for daily nutrition and industrial bioproduction of mushroom mycelia enriched with selenium.