Wastewater from the textile industry contaminated with azo dyes affects the environment negatively, causes pollution, and threatens environmental balance. Among various methods for wastewater treatment, bioremediation emerges as an environmentally friendly, economical, and sustainable solution. In this study, white-rot fungus Sporotrichum sp. was employed to decolorize reactive blue 13 (RB13). The long-term decolorization capacity of the fungus was investigated by a sequential batch experiment under optimized conditions. The fungus showed high decolorization efficiency upon repeating usage, and its decolorization efficiency decreased from 97.4% to 87.09% after transferring to a freshly prepared medium seven times. The MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay) method using Chinese Hamster Lung V79 379A was performed to assess the cytotoxicity of treated water samples. This study revealed that Sporotrichum sp. has short-term enzymatic and long-term biosorption capacity on reactive blue 13 and the decolorization potential of the alive and dead cells is impressively high. Practitioner Points White-rot fungus Sporotrichum sp. is able to decolorize sulfonated azo-dye reactive blue 13 upon sequential incubation in freshly prepared dye solution. The decolorization mechanism of the fungus is estimated to be bioadsorption. Sporotrichum sp. can be considered for long-term usage and immobilization applications.