Electron beam irradiation (EBI) has been considered an advanced technology for the treatment of water and wastewater, whereas very few previous investigations reported its use for removing pharmaceutical pollutants. In this study, the degradation of fluoxetine (FLX), an antidepressant marketed as Prozac(®), was investigated by using EBI at FLX initial concentration of 19.4 ± 0.2 mg L(-1). More than 90 % FLX degradation was achieved at 0.5 kGy, with FLX below the detection limit (0.012 mg L(-1)) at doses higher than 2.5 kGy. The elucidation of organic byproducts performed using direct injection mass spectrometry, along with the results of ion chromatography, indicated hydroxylation of FLX molecules with release of fluoride and nitrate anions. Nevertheless, about 80 % of the total organic carbon concentration remained even for 7.5 kGy or higher doses. The decreases in acute toxicity achieved 86.8 and 9.6 % for Daphnia similis and Vibrio fischeri after EBI exposure at 5 kGy, respectively. These results suggest that EBI could be an alternative to eliminate FLX and to decrease residual toxicity from wastewater generated in pharmaceutical formulation facilities, although further investigation is needed for correlating the FLX degradation mechanism with the toxicity results.