A variety of white-rot fungi can oxidize textile dyes under sterile conditions; however, an important consideration for their use in treating wastewater containing textile dyes is whether similar degrees of treatment can be achieved under non-sterile conditions. Four strategies were investigated for their potential in optimizing the use of the fungus Trametes versicolor in non-sterile culture for treating wastewater containing the diazo textile dye C.I. Reactive Black 5 (RB5). Three strategies with suspended culture were designed to increase the decolorization activity in suspended culture from a given amount of T. versicolor inoculum based on its tolerance of low pH (pH reduction in medium), production of extracellular enzymes (use of suspended enzymes alone), and its ability to produce enzymes independent of growth (nitrogen limitation in medium). The results showed that reduction of the medium pH to 3 did not suppress bacterial growth, while enzyme production by T. versicolor ceased. The use of the extracellular enzymes alone would allow the decoupling of the process of fungal growth from wastewater treatment; however, the enzyme activity of an enzyme suspension decreased rapidly under non-sterile conditions. The strategy of limiting nitrogen in the medium to suppress bacterial growth has potential together with the fourth strategy, the cultivation of fungi on organic solids to produce inocula for a decolorization process under non-sterile conditions. A high degree of decolorization of RB5 under non-sterile conditions was achieved with T. versicolor grown on grains as sole substrate. The rate of decolorization was dependent on the amount of fungal inoculum used.