The agricultural residues are ecofriendly alternatives for removing contaminants from water. In this way, a novel biochar from the spent mushroom substrate (SMS) was produced and assessed to remove endocrine disruptor from water in batch and fixed-bed method. SMS were dried, ground, and pyrolyzed. Pyrolysis was carried out in three different conditions at 250 and 450 °C, with a residence time of 1 h, and at 600 °C with a residence time of 20 min. The biochar was firstly tested in a pilot batch with 17α-ethinylestradiol (EE2) and progesterone. The residual concentrations of the endocrine disruptors were determined by HPLC. The biochar obtained at 600 °C showed the best removal efficiency results. Then, adsorption parameters (isotherm and kinetics), fixed bed tests and biochar characterization were carried out. The Langmuir model fits better to progesterone while the Freundlich model fits better to EE2. The Langmuir model isotherm indicated a maximum adsorption capacity of 232.64 mg progesterone/g biochar, and 138.98 mg EE2/g biochar. Images from scanning electrons microscopy showed that the 600 °C biochar presented higher porosity than others. In the fixed bed test the removal capacity was more than 80% for both endocrine disruptors. Thus, the biochar showed a good and viable option for removal of contaminants, such as hormones.