Polycyclic Aromatic Hydrocarbons (PAHs) are complex compounds generated from industrial and anthropogenic activities, although natural phenomena are also responsible for their presence in the environment. The aim of this study was to evaluate the effect of phenanthrene on proximate composition and antioxidant activity in Piaractus brachypomus during a subacute exposure. Fish were exposed intraperitoneally to phenanthrene (0.1, 1.0, and 10 μg g-1) and a solvent control (0 μg g-1). Muscle tissue, liver and bile were collected at 0 h, 11 days and 21 days. In dorsal muscle, parameters such as dry matter, protein, crude fat, and pH did not show alterations with exposure to phenanthrene (p ≥ 0.05). At 11 days, a significant increase of hepatic lipid peroxidation was observed in fish exposed to 10 μg g-1. Additionally, a dose-dependent response was observed in the muscle, although no significant differences were observed in the activity of catalase and lipid peroxidation. Phenanthrene metabolites in bile were analyzed by fixed fluorescence at 260/380 nm (excitation/emission) wavelengths and levels indicated a dose-dependent response. Likewise, bioaccumulation of phenanthrene in bile was observed for 21 days, suggesting slow metabolism of this xenobiotic at 10 μg g-1. This study provides important information on the oxidative effects generated by phenanthrene exposure for 21 days in P. brachypomus, generating fundamental information on adverse effects induced by PAHs on the most important Colombian native fish species. These data contribute to the development of additional research in environments contaminated with PAHs and can be applied to monitor areas contaminated with oil spills.