A cell immobilization technique using polytetrafluoroethylene (PTFE) was employed in biotransformation process. Conversion of methyltestosterone to methandienone was achieved using the immobilized cells. An optimization strategy included two levels factorial model and three levels model was used. The most significant factors, glucose concentration, shaking speed and reaction time, were determined after Plakett-Burman screening study. Thereafter, the three levels quadratic model determined the optimum measures for thses significant factors and predicted the maximum product yield. The optimization led to improvement of the methandienone yield, from 45 % to 85% using the immobilized cells. The immobilized cells were reused for six runs with a good conversion outcomes and retained its metabolic activities after storage at 4ºC for one month. To the best of our knowledge, no previous work on this topic, the optimization and production of methandienone using the immobilized cells. The isolated fungus, Circinella mucoroides, was not previously used in similar work.