Ascochlorin is a potent antiviral, antitumor and breast cancer suppressor compound. The optimization of the fermentation conditions for the production of this compound by Microcera sp. BCC 17074 was conducted using a multi-step strategy. The first step was the selection of carbon and nitrogen sources using a full factorial design. This was followed by a Plackett-Burman design to select the most important factors among the nine medium components and a central composite design to determine the optimal five key components. The utility of the optimized medium was confirmed in a 5-L bioreactor. Fructose and yeast extract were the most preferable carbon and nitrogen sources, respectively. The results of the Plackett-Burman design yielded a medium containing 60 g/L fructose, 6 g/L yeast extract, 2 g/L NaCl, 0.2 g/L CaCl₂.2H2O and 2 g/L K₂HPO₄. Evaluating the levels of the main factors using the central composite design, an ascochlorin production of 41.12±0.62 mg/L was reached with a medium containing 40 g/L fructose, 4 g/L yeast extract, 3 g/L NH4Cl, 3 g/L NaCl and 4 g/L KH₂PO₄. The chloride ions drastically affected the ascochlorin production. The production of ascochlorin in a 5-L bioreactor gave an ascochlorin production of 68.35±1.17 mg/L at 144 h and a highest biomass production of 19.6±0.99 g/L at 193 h. These results demonstrated that Microcera sp. BCC 17074 serves as a useful cell factory for ascochlorin production.