Central composite experimental design was employed to determine the optimal concentration of medium constituents for griseofulvin production by Penicillium griseofulvum MTCC 1898 and Penicillium griseofulvum MTCC 2004. The optimal concentration of sucrose, K2HPO4, NaNO3, and FeSO4.7H2O were found to be 48.08 g/l, 1.228 g/l, 2.7 g/l, and 0.011 g/l, respectively, for Penicillium griseofulvum MTCC 1898, and for Penicillium griseofulvum MTCC 2004, 23.52 g/l, 43.67 g/l, and 0.0434 g/l of glucose, lactose, and MnSO4·H2O, respectively. The yield of griseofulvin under optimal composition of medium constituents increased by 1.26 and 1.38 times than prior to optimization, for Penicillium griseofulvum MTCC 1898 and Penicillium griseofulvum MTCC 2004, respectively.