Decolourization and degradation of the diazo dye Reactive Black 5 was carried out by the yeast Trichosporon akiyoshidainum. A nine-factor Plackett-Burman design was employed for the study and optimization of the decolourization process and production of manganese peroxidase (MnP) and tyrosinase activities. In the present study, 26 individual experiments were conducted and three responses were evaluated. Raising yeast extract concentration significantly enhanced decolourization and MnP production. Carbon and nitrogen sources, glucose and (NH 4 ) 2 SO 4 , showed no significant effect on any response over the concentration range tested. Other culture medium components, such as CaCl 2 or MgSO 4 , could be excluded from the medium formula, as they had no effect on the evaluated responses. Metal ions (Fe, Cu and Mn) showed different effects on decolourization and enzymatic activities. Addition of copper significantly enhanced MnP activity and decreased dye decolourization. On the contrary, iron had a positive effect on decolourization and no effect on enzyme production. Oddly, increasing manganese concentration had a positive effect on tyrosinase production without affecting decolourization or MnP activity. These results strongly suggest that dye decolourization should be regarded as a complex multi-enzymatic process, where optimal medium composition should arise as a compromise between those optimal for each implied enzyme production.