The oxidant Mn3+-malonate, generated by the ligninolytic enzyme versatile peroxidase in a two-stage system, was used for the continuous removal of endocrine disrupting compounds (EDCs) from synthetic and real wastewaters. One plasticizer (bisphenol-A), one bactericide (triclosan) and three estrogenic compounds (estrone, 17-estradiol, and 17-ethinylestradiol) were removed from wastewater at degradation rates in the range of 28-58 mu g/Lmin, with low enzyme inactivation. First, the optimization of three main parameters affecting the generation of Mn3+-malonate (hydraulic retention time as well as Na-malonate and H2O2 feeding rates) was conducted following a response surface methodology (RSM). Under optimal conditions, the degradation of the EDCs was proven at high (1.3-8.8 mg/L) and environmental (1.2-6.1 mu g/L) concentrations. Finally, when the two-stage system was compared with a conventional enzymatic membrane reactor (EMR) using the same enzyme, a 14-fold increase of the removal efficiency was observed. At the same time, operational problems found during EDCs removal in the EMR system (e.g., clogging of the membrane and enzyme inactivation) were avoided by physically separating the stages of complex formation and pollutant oxidation, allowing the system to be operated for a longer period (approximate to 8 h). This study demonstrates the feasibility of the two-stage enzymatic system for removing EDCs both at high and environmental concentrations. (c) 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:908-916, 2015