In this study, we investigated the effect of repeated handling on the oxidative state of gilthead sea bream, Sparus aurata. We determined the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPX), glutathione transferase (GST), and DT-diaphorase (DTD) in liver, digestive tract, brain, gills, and white muscle. We also measured the lipid-peroxidation level (malondialdehyde – MDA – concentration) and the glucose 6-phosphate dehydrogenase activity (G6PDH). The results show that repeated low-intensity handling on gilthead sea bream alter the oxidative status of tissues such as liver, digestive tract, and muscle in the last term can affect its welfare. The brain in a situation of handling stress is protected from oxidative damage by the activity of antioxidant enzymes. Finally, the concentration of MDA in tissues can be a good candidate as a biomarker of stress and welfare in fish.