Penicillium sp. (IBWF 040-09) produces a protease inhibitor that can potentially be used against the main protease of human African trypanosomiasis. Since the target substance is formed intracellularly (under nutrient limitation), the fungal pellet is preferred compared to the free mycelia in bioreactor cultivation. The optimization of the production of protease inhibitor became the main focus of this study. The effects of the concentrations of spores, calcium chloride, and Pluronic F68 were investigated with regard to fungal growth, pellet morphology, and the production of protease inhibitor. The combination of adjusting the spore concentration and adding Pluronic F68 and calcium chloride increased the probability of achieving the desired morphology. This ensured better reproducibility of the production of the target substance by Penicillium sp. (IBWF 040-09) with the bioreactor system used. In addition, the protease inhibitor was tested in a resazurin assay and showed no noticeable cytotoxic effects on peripheral blood mononuclear cells isolated from whole blood cells.