Sulfamethoxazole (SMX) is a synthetic antibiotic widely applied as a bacteriostatic drug to treat a number of diseases. SMX can persist in the environment for long periods of time because of its low biodegradability, which may result in various, direct and indirect, toxicological effects on the environment and on human health. Therefore, we have developed the electrochemical advanced oxidation process (AOP) "electro-Fenton" to degrade SMX in aqueous media. In this work, a detailed study of the evolution of toxicity of SMX and its degradation products in aqueous solutions, during treatment by the electro-Fenton AOP, is described, using the bioluminescence Microtox® method, based on the inhibition of luminescence of marine bacteria Vibrio fischeri. Samples were collected at various electrolysis times and analyzed by HPLC for quantifying the evolution of the degradation products, and their toxicity was measured by the Microtox® method. Our results demonstrated that the toxicity of SMX aqueous solutions varied considerably with the electrolysis time and the applied current intensity. This phenomenon could be explained by the formation and disappearance of several degradation products, including cyclic and/or aromatic intermediates, and short-chain acid carboxylic acids, having a toxicity different of the initial antibiotic. The curves of the % of bacterial luminescence inhibition vs. electrolysis time, corresponding to the evolution of the toxicity of the formed degradation products, were investigated and tentatively interpreted.