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The aim of this work was to optimize the production of a novel antimaralial menisporopsin A by the seed fungus Menisporopsis theobromae BCC 4162.Fungal cultures were grown in shake flasks at 25°C in the basal medium with varying carbon and nitrogen sources, aeration rates and initial pH levels. The optimal carbon and nitrogen sources that improved the production of menisporopsin A were 1% fructose and 2·5% meat extract respectively. The production was further enhanced when the culture incubated on a shaker at 200 rev min−1 with an initial pH of 8. The yield of menisporopsin A cultured under the optimized conditions was increased from 348·30 (obtained from basal medium) to 889·02 mg l−1, and the cultivation time was reduced from 28 to only 4 days. As a result, the productivity of menisporopsin A was greatly enhanced to 222·26 mg l−1 day−1 which is 18-fold higher than that of basal conditions. Larger scale production in a fermenter was also achieved, yielding menisporopsin A at a maximal level of 594·32 mg l−1 in 4 days.The optimized culture conditions for menisporopsin A production by M. theobromae BCC 4162 was the cultivation under shaking or agitation at 25°C in fructose–meat extract medium with an initial pH of 8.The production of menisporopsin A in a fermenter with a relatively short incubation period could be valuable for further utilization for chemical structure modification and derivatization.