Aims: This work aimed to optimize the culture conditions for production of a novel and potent anti-tubercular alkaloid, hirsutellone A, by the saprophytic soil fungus Trichoderma gelatinosum BCC 7579. Methods and Results: The fungus was initially cultured in shake flasks at 25°C in the potato dextrose broth (PDB) supplemented with various carbon and nitrogen sources and mineral salts to select suitable medium for mycelial growth and hirsutellone A production. Cultivation conditions were further optimized by adjusting initial pH and changing temperature levels to maximize the production of hirsutellone A. The optimal condition that increased the production of hirsutellone A from 19.04 mg l-1, obtained from basal condition, to 610.55 mg l-1 and reduced the cultivation time from 40 to 6 days was to cultivate in a shaker at 200 rev min-1 at 25°C in PDB plus 20 g l-1 soluble starch, 10 g l-1 peptone and 2.5% (v/v) salt solution with initial pH of 7. Production of hirsutellone A in larger-scale using a 5-l batch fermenter was also completed yielding 958 mg l-1 of hirsutellone A within 6 days. Conclusions: The suitable culture conditions for hirsutellone A production by T. gelatinosum BCC 7579 was the cultivation in 5-l fermenter at 25°C in PDB plus 20 g l-1 soluble starch, 10 g l-1 peptone and 2.5% (v/v) salt solution with an initial pH of 7. Significance and Impact of the Study: The production of hirsutellone A in a fermenter to obtain a high yield and reduce an incubation period will become very useful in anti-tubercular drug development process in the future. © 2007 The Authors.