This study demonstrates the effect of cultivation conditions on the production of natamycin. Of these conditions, the effect of oxygen limitation and type of inoculum were extensively investigated. Increasing the shaking speed and decreasing the medium volume improved both the volumetric and specific natamycin production. Also, decreasing the dissolved oxygen level in the cultivation medium through the addition of soluble biopolymer (alginate) resulted in a significant decrease in the natamycin yield without effect on the cell growth. On the other hand, spore inoculum yielded higher concentration of natamycin compared to the vegetative cells by about 40%. The maximal cell productivity based on biomass [Yp/x] of about 0.4 [g/g] was obtained by using shake flask of 50 ml working volume agitated at 200 rpm and the inoculum was in the form of spore 2 × 108 spores/ml. These results showed that the production process of natamycin is highly dependent on oxygen level in the cultivation medium and type of inoculum as well.