Abstract: The combined effects of macronutrients of media on glucoamylase production by Candida guilliermendii were studied using Design Of Experiment (DOE). A 2P-k factorial design was chosen to explain fifteen medium constituents: pH, Starch, Sucrose, Yeast extract, Peptone, NH4Cl (NH4)2HPO4, NH4NO3, CH4NO2 (NH4)2SO4, CaCl2, MnCl2, FeCl2, ZnCl2 and MgCl2 and analyse the results. This procedure limited the number of actual experiments performed while allowing for possible interactions between components. The p-value of the coefficient for quadratic effect of pH, starch and yeast extract concentration was < 0.001, suggesting that they were the main experimental variables having the highest effect on the production of glucoamylase. It was found that yeast extract had a great effect on glucoamylase production. The optimal combinations of media constituents for maximum which were chosen for further studies on production of glucoamylase were determined as 10 g L-1 starch, 0.45 g L-1 urea, 0.61 g L-1 NH4NO3, 3 g L-1 Yeast extract and 0.1 g L-1 Mg SO4.