Taxol is the most profitable drug ever developed in cancer chemotherapy; however, the market demand for the drug greatly exceeds the supply that can be sustained from its natural sources. In this study, Aspergillus fumigatus TXD105–GM6 and Alternaria tenuissima TER995–GM3 were immobilized in calcium alginate beads and used for the production of taxol in shake flask cultures. In an effort to increase the taxol magnitude, immobilization conditions were optimized by response surface methodology program (RSM). The optimum levels of alginate concentration, calcium chloride concentration, and mycelium fresh weight were 5%, 4%, and 15% (w/v), respectively. Under these conditions, taxol production by the respective fungal strains was intensified to 901.94 μg L−1 and 529.01 μg L−1. Moreover, the immobilized mycelia of both strains were successfully used in the repeated production of taxol for six different fermentation cycles. The total taxol concentration obtained in all cycles reached 4540.14 μg L−1 by TXD105–GM6 and 2450.27 μg L−1 by TER995–GM3 strain, which represents 7.85- and 6.31-fold increase, as compared to their initial titers. This is the first report on the production of taxol in semi-continuous fermentation. To our knowledge, the taxol productivity achieved in this study is the highest reported by academic laboratories for microbial cultures which indicates the future possibility to reduce the cost of taxol production.