We investigated the effect of nutritional and environmental factors on Ophiocordyceps longissima mycelial growth. The longest colony diameter was observed on Schizophyllum (mushroom) genetics complete medium plus yeast extract, Schizophyllum (mushroom) genetics minimal medium, and Sabouraud dextrose agar (SDA); however, malt-extract yeast-extract agar, SDA plus yeast extract, yeast-extract malt-extract peptone dextrose agar, SDA, oatmeal agar, and potato dextrose agar showed higher mycelia density. A temperature of $25^{circ}C$ was optimum and 7.0 was the optimum pH for mycelial growth. Colony diameter was similar under light and dark conditions. Maltose and yeast extract showed the highest mycelial growth among carbon and nitrogen sources respectively. The effect of mineral salts was less obvious; however, $K_3PO_4$ showed slightly better growth than that of the other mineral salts tested. Among all nutrition sources tested, complex organic nitrogen sources such as yeast extract, peptone, and tryptone were best for mycelial growth of O. longissima. Ophiocordyceps longissima composite medium, formulated by adding maltose (2% w/v), yeast extract (1% w/v), and $K_3PO_4$ (0.05% w/v) resulted in slightly longer colony diameter. In vitro mycelial O. longissima growth was sustainable and the production of fruiting bodies could be used for commercial purposes in the future.