THE PRODUCTION of bioactive compounds by endophytic Chaetosphaeronema sp. (KY321184) has been optimized using the experimental design methods. Among seven fermentation parameters, Plackett-Burman design revealed that potato extract concentration, glucose concentration and inoculum size are three significant variables positively affecting the production process. The levels of these variables have been optimized using Box-Behnken design. The best productivity was estimated under potato extract concentration, glucose concentration and inoculum size of 190gL-1, 15gL-1 and 5 fungal discs (0.8cm diameter), respectively. The model was validated experimentally, and it showed a deviation error of 4.3% and 5.3% from the predicted values of Design Expert's model and the Excel solver, respectively, which considered as an acceptable error. Moreover, it has been observed that, trails of Plackett-Burman design stimulated the production of different compounds and one of them is produced in a partially pure form. This observation suggests that, this type of experimental design not only can be employed for optimization purpose but also may represent a method to modify the production pattern of bioactive compounds in a trial to obtain them in a partially pure form to save the cost and effort exerted in the purification process.