This study was conducted to investigate a suitable substrate formulation for bag cultivation of Hypsizygus marmoreus. To determine the optimum media composition for H. marmoreus, chemical properties of various media and culture properties were investigated. The fastest primordia formation (9.0 days), the shortest fruiting body harvest period (18.0 days), and the highest yield (144.7 g/bag) were realized with the T6 formulation (55:15:20:5:5 mixing ratio of poplar sawdust, wheat bran, rice bran, dried soybean powder, and palm kernel, respectively). The chemical properties of the T6 formulation were pH, 6.0; total carbon, 19.0%; total nitrogen, 0.83%; C/N ratio, 22.8; P2O5, 0.61%; K2O, 0.46%; and MgO, 0.29%. This is the first attempt using plastic bags for cultivation and will help expand the production of H. marmoreus.