The production of antibiotics in Algeria generates large amounts of residual biomass that should be considered a valuable biosorbent rather than waste. The aim of this work was to assess the use of antibiotic by-product biomass as a biosorbent for methylene blue removal from wastewater. Therefore, the biosorption of the cationic dye methylene blue by spent biomass of Pleurotus mutilus immobilized as calcium alginate biobeads (CAB) was studied in batch and column modes. The maximum uptake capacity was found to be 23 mg/g at a pH value of 10. Methylene blue biosorption by CAB was found to be spontaneous and exothermic. The adsorption capacity of the used biobeads was regenerated in continuous mode by washing the CAB with HCl (0.5 M) solution. Globally, this work showed the high potential of CAB made with spent biomass of P. mutilus as a biosorbent for the treatment of water contaminated with methylene blue.