To determine a favorable substrate formulation for Grifola frondosa, physicochemical conditions, culture properties, and yields according to various substrate formulations were investigated. Based on these analyses, T4 (80:5:15 ratio of oak sawdust to dried bean-curd refuse to corn husk) resulted in a shorter cultivation period and higher yields (weight of fresh mushrooms harvested at maturity) than those of other treatments. The physicochemical properties of T4 were pH 5.4, 2.4% crude fat contents, 54 C/N ratio, 74.3% porosity, and 0.26 g/cm3 bulk density. These results emphasize the importance of optimal substrate development on the production efficiency of G. frondosa mushrooms and have implications for commercial applications.