Investigation and optimization of culturing conditions were performed for the production of microbial surfactant, O-acid (precursor of S-acid) using Pencilium spiculisporum. Glucose and ammonium nitrate were found to be the most effective carbon and nitrogen sources, respectively. Supplementation of medium with trace elements, such as $CaCl_2 and FeSO_4$, increased the O-acid production by 20% and maintenance of the dissolved oxygen tension near saturation increased 40% of the O-acid productivity. Also 60% increase in the O-acid production was observed by maintaining the glucose concentration near 50%g/l by feeding glucose during the cultivation.