The aim of the present work is to investigate the removal and transformation of the antidepressants venlafaxine (VFX) and its main metabolite O-desmethylvenlafaxine (DVFX) upon advanced oxidation with UV/H2O2 under lab conditions. High-resolution mass spectrometry (HRMS) analyses were carried out by means of ultra-high pressure liquid chromatography (UHPLC)-linear ion trap high resolution Orbitrap instrument (LTQ-Orbitrap-MS) in order to elucidate the different transformation products (TPs) generated. The depletion of both VFX and DVFX was very significant, with the 99.9% of both compounds eliminated after 5 and 30 min of reaction, respectively. Eleven TPs for VFX and six for DVFX were detected and their molecular structures elucidated by means of MS2 and MS3 scans, and the corresponding degradation pathways were proposed. The combined ecotoxicity at different treatment times was evaluated by means of bioluminescence inhibition assays with the marine bacteria Vibrio fischeri. Results showed an increase in the ecotoxicity during the UV/H2O2 experiment, especially at those reaction times where the total abundance of TPs was higher.