Ecumicin is a novel anti-tuberculosis agent produced by Nonomuraea sp. MJM5123 as a new strain of actinomycetes. First, in order to increase the cell mass of Nonomuraea sp. MJM5123, we optimized the culture conditions with regard to carbon and nitrogen sources. The cell mass of Nonomuraea sp. MJM5123 increased by approximately twofold when glucose and soybean flour were used as carbon and nitrogen sources, respectively. For maximum production of ecumicin, we optimized the culture conditions by adding amino acids as building blocks for ecumicin, by adding vegetable oils and by controlling the temperature and pH. Ecumicin production was two times higher with the addition of valine as the building blocks for ecumicin compared with the production in the absence of valine. Interestingly, with the addition of 1% corn oil, the production of ecumicin increased by 4.6-fold compared with the production in the absence of corn oil. Finally, by controlling the pH and temperature, we established an optimized culture condition in which Nonomuraea sp. MJM5123 produced 576 mg ecumicin per litre of medium, which is about 50 times higher than in the control medium at 30 °C and pH 7.0.