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The aim of this study was to evaluate the biodecolorization and detoxification of the anticancer drug mitoxantron (MTX) by immobilized crude versatile peroxidase of <i>Bjerkandera adusta</i> CCBAS 930 (icVP/Ba). The concentrated crude VP was obtained from <i>B. adusta</i> CCBAS 930 culture on medium with MTX (µg/mL) addition, immobilized with 4% sodium alginate. MTX removal degree (decolorization), levels of phenolic compounds and free radicals were determined during MTX biotransformation. Moreover, the phytotoxicity (<i>Lepidium sativum</i> L.), biotoxicity (multi-species microbial assay, MARA), and genotoxicity (SOS Chromotest) of MTX were evaluated before and after the biological treatment. The use of icVP/Ba (95 U/mL) significantly shortened the bioremoval of 10 µg/mL MTX (95.57% after 72 h). MTX removal by icVP/Ba was correlated with an 85% and 90% decrease in the levels of phenolic compounds and free radicals, respectively. In addition, the use of icVP/Ba contributed to a decrease in the phyto-, bio-, and genotoxicity of MTX. This is the first study to describe the possibility of removing MTX using immobilized crude fungal peroxidase.