The methods for increasing the production of ergothioneine (ERG) were investigated by using the mycelial culture of several mushroom species, primarily Ganoderma neo-japonicum. We first found that ERG was accumulated at the different levels in mycelia and fruiting bodies, respectively, depending on the mushroom species. As a result of adding various amino acids to the mycelial culture medium, methionine (Met) was shown to be the most effective additive. The most preferable condition of the additive was the combination of 4 mM Met and 1 g/l of yeast extract, and the maximum ERG production reached approximately 1.7 mg/l, which corresponds to 2.4 times (0.7 mg/l) that in the basal medium without Met. Although the supplementation of Met enhanced the ERG production, the mycelial growth was significantly inhibited. Furthermore, the analysis of amino acids in the culture medium revealed that the Met additive reduced the consumption rates of most amino acids tested, probably due to the decrease in mycelial growth. Taking these results into consideration, we suggest that the addition of Met to the mycelial culture medium is an efficient way to enhance the ERG production in economically important mushroom species.