Mycoepoxydiene exhibited broad spectrum of cytotoxicity against human tumor cell lines. According to the interesting cytotoxic activities of this compound, its mechanism of action and related studies should be pursued, and these experiments require a large amount of mycoepoxydiene. This study aims to improve mycoepoxydiene yield by varying fermentation conditions of Phomopsis sp. Hant25, an endophytic fungus isolated from Hydnocarpus anthelminthicus. Agar diffusion assay to determine relative amounts of mycoepoxydiene produced by different fermentation conditions was developed. Mycoepoxydiene was found to be clearly active against Candida albicans when tested in medium containing 12.5 µg/mL of ketoconazole. Several fermentation conditions were conducted. Culture media affected mycoepoxydiene production by the fungus. Mycoepoxydiene obtained from conventional shaken and stationary liquid fermentations were low. Analysis by HPLC revealed that mycoepoxydiene amount was increased approximately 13.56 times yielding 333 mg/L when cultured in MM1D medium with filter support at 25°C under static condition for 6 days and then agitation at 120 rpm for 12 days. Fungal morphology in culture medium during fermentation was found to be related to mycoepoxydiene yield.