Mycobacterium spp. was used for biotransformation of phytosterol to androstenedione. The study signifies the importance of media design and that medium constituents play a crucial role in the biotransformation process. Various statistical experiments namely, categorical, numerical factorial design and central composite design were applied to optimize the medium. The carbon and nitrogen sources, emulsifier source and the type of phytosterol were screened through categorical design. The significant components among the four were analyzed through full factorial design. Based on the results obtained, the optimal concentration of the significant components was determined using central composite design. With the tween 80, soya oil and phytosterol concentration to be 15 g/l, 25 g/l and 40 g/l there was a 50% enhancement in the yield of androstenedione. The methods used were very effective in screening the significant components in a limited number of experiments.