The Plackett-Burman experimental design was employed to evaluate the relative importance of medium constituents of each medium for enhanced griseofulvin production by Penicillium griseofulvum MTCC 1898 and Penicillium griseofulvum MTCC 2004. It was found that the medium constituents, sucrose, K2HPO4, NaNO3, and FeSO4.7H2O, significantly influenced the griseofulvin production by Penicillium griseofulvum MTCC 1898. In the case of Penicillium griseofulvum MTCC 2004, lactose, glucose, and MnSO4·H2O significantly influenced the griseofulvin production.