One of the main challenges in both the design of new wastewater treatment plants and the expansion and improvement of existing ones is the removal of emerging pollutants. Therefore, the search for economic and sustainable treatments is needed to enhance the removal of pharmaceuticals. The potential of a lignocellulosic substrate colonized by Pleurotus ostreatus, a waste from mushroom production, to remove fluoxetine from aqueous solutions was studied. Batch assays were performed to remove 600 µg∙L−1 fluoxetine from aqueous solutions using the colonized mushroom substrate (CMS) and crude enzyme extracts. The removal efficiencies achieved were, respectively, ≥83.1% and 19.6% in 10 min. Batch assays with sterilized CMS and 1-aminobenzotriazole (to inhibit cytochrome P450 enzymes) showed that the higher removal efficiencies achieved in the CMS assays may be attributed to the synergistic contribution of biosorption onto the CMS and lignin modifying enzymes activity, namely laccase activity. A column assay was performed with the CMS, fed with 750 µg∙L−1 fluoxetine aqueous solution. The removal efficiency was 100% during 30 min, decreasing to a final value of 70% after 8 h of operation. The results suggested that CMS can be a promising eco-friendly alternative to remove fluoxetine from aqueous solutions.