Decolourization of Mordant Blue-9 (MB-9) by the white-rot fungi Phanerochaete chrysosporium MTCC 787 was investigated by screening and optimization of the media constituents used for growing the fungus. Both % decolourization and specific removal of MB-9 by the fungus were taken as the responses to screen and optimize the media constituents using statistically valid Plackett–Burman and response surface methodology (RSM) design of experiments, respectively. Amongst the media constituents screened, glucose, veratryl alcohol, KH₂PO₄ and CaCl₂ were selected as the most important (P value < 0.05) media constituents for MB-9 dye decolourization; the other media constituents, viz. MgSO₄, tween 20, NH₄Cl₂ and inoculum size were, however, found to be insignificant in the study. Central composite design followed by RSM used in optimizing the important media constituents for enhancing the decolourization of MB-9 dye revealed optimum combinations of glucose, 13.46 g l⁻¹; veratryl alcohol, 9.30 mM; KH₂PO₄, 24.52 g l⁻¹; CaCl₂, 2.18 g l⁻¹; MgSO₄, 9.89 g l⁻¹; NH₄Cl₂, 4.68 g l⁻¹; tween 20, 0.050% and inoculum size, 0.8 OD₆₅₀, which gave a maximum % dye decolourization of 100% and specific dye removal of 0.1571 mg U⁻¹.