To determine the optimum amount of spent oyster mushroom substrate (SOMS) for use in cultivation of Lentinula edodes, the chemical properties of the substrate and culture conditions of Lentinula edodes were investigated. Replacing 20–50% of a sawdust substrate with SOMS yielded a C/N ratio of 62–76. In case of substrates containing SOMS, the total nitrogen and phenolic contents of were higher, whereas fructose and organic acid contents were lower than those of the control substrate. Cultivation tests showed that the 3-cycle yield of 20% SOMS treatment was 286.7 g, similar to that of the control, while 50% SOMS treatment significantly decreased the yield. In conclusion, development of oak mushroom substrate using SOMS would recycle waste products and decrease material costs.