This study was conducted to investigate the optimum additive ratio of barley flour when used instead of cotton seed meal for the cultivation of oyster mushrooms. Mycelial growth was the fastest to 11.4 cm in medium containing 5% barley flour and to 9.0 cm in medium containing 30% barley flour. The mycelial density was slightly lower at 5% and 10% of barley powder addition, but there was no significant difference between the other treatments. The yield of the fruiting bodies was 158 g/850 mL, which was highest on treatment with 10% barley flour as a substitute for cotton seed meal. However, this was lower on treatments with only barley flour than in the control (156 g/850 mL). The diameter of the pileus and the hardness of stipes were highest with 10% barley flour and 10% in cotton seed meal, respectively. The L value was highest with 10% added barley flour, but the a-value and the b-value were not significantly different between treatments. The pH of the mixed media before inoculation with Pleurotus ostreatus was 4.7–5.0, and the total nitrogen content was 1.7–2.2, which was slightly lower than that of the control. The total carbon content was 44.6–45.0, which was not significantly different from that of the control. However, the content of trace elements such as Cu and Fe was lower than those of the control.