Because of its effectiveness in organic pollutant degradation, manganese peroxidase (MnP) enzyme has attracted significant attention in recent years regarding its use for wastewater treatment. Herein, MnP was extracted from Anthracophyllum discolor fungi and immobilized on the surface of magnetic nanocomposite Fe3O4/chitosan. The prepared nanocomposite offered a high surface area for MnP immobilization. The influence of several environmental factors like temperature, pH, as well as storage duration on the activity of the extracted enzyme has been studied. Fourier transmission infrared spectroscopy (FT-IR), scanning electron microscope (SEM), X-ray diffraction (XRD), and transmission electron microscope (TEM) techniques were used for the characterization of the prepared MnP/Fe3O4/chitosan nanocomposite. The efficiencies of the prepared MnP/Fe3O4/chitosan nanocomposite for the elimination of reactive orange 16 (RO 16) and methylene blue (MB) industrial dyes were determined. According to the results, the immobilization of MnP on Fe3O4/chitosan nanocomposite increases its capacity to decolorize MB and RO 16. This nanocomposite allowed the removal of 96% ± 2% and 98% ± 2% of MB and RO 16, respectively. The reusability of the synthesized nanocomposite was studied for five successive cycles showing the ability to retain its efficiency even after five cycles. Thus, the prepared MnP/Fe3O4/chitosan nanocomposite has potential to be a promising material for textile wastewater bioremediation.