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<i>Ganoderma formosanum</i> is a medicinal mushroom endemic to Taiwan. The extraction of <i>G. formosanum</i> mycelium using ethyl acetate showed a significant reduction of melanin activity due to the inhibition of tyrosinase. To optimize the production of <i>G. formosanum</i> mycelium with anti-melanogenic activity, different variables (carbon sources and concentrations, initial pH value, and temperature) were investigated. According to the results, the optimal conditions included the utilization of 50 g/L lactose as the carbon source at pH 7 and 25 °C for 9 days. Afterward, the <i>G. formosanum</i> ethanolic extracts-optimized (GFE-O) was used to study the anti-melanogenic activity in vitro and in vivo. In B16-F10 melanoma cells treated with GFE-O (0.1 mg/mL), the intracellular melanin content was reduced to 76% compared to the control group. By applying GFE-O (0.05 mg/mL) in vivo on zebrafish embryo, hypopigmentation was observed, and the melanin content was reduced to 62% compared to the control with no toxicological effects. The results showed that the optimal inoculation conditions can provide the basis for future large-scale production of <i>G. formosanum</i> mycelium to maximize the extraction of anti-melanogenic ingredients.