The evaluation of antioxidant system capacity is important in aquatic toxicology. It was aimed to characterize the liver antioxidant enzymes (SOD, CAT, GPX, GR, and GST) and to test the in vitro Zn effect (200 and 400 ZnSO4 μg/L) in native fish Barbus meridionalis obtained from the Osor River (NE, Spain) influenced by Zn contamination. The maximal enzyme activities were at pH 7.0–7.5 and 100 mM phosphate buffer. Barbel showed high catalytic activity (high Vmax and low Km) indicating the efficient antioxidant detoxification ability. Direct Zn effect caused an antioxidant system imbalance. Mostly upon lower Zn concentration, GPX activity decreased (95–100 %) though CAT, GR, and GST increased (36–1543 %). GSH values either stimulated (290 %) or inhibited (85–93 %) due to tissue differences. The first record of barbel antioxidant enzyme characterization and in vitro data presenting an unbalanced antioxidant pattern could be significant to evaluate the metal pollution in the Osor River for further in vivo studies.