The present study aimed to evaluate the biological responses of Colossoma macropomum to naphthalene injection and subsequent hypoxia exposure, emphasizing the expression of the tumor suppressor gene tp53. Tambaquis were intraperitoneally injected with naphthalene (50 mg/kg) and, after 96 hours, the fish were transferred to respirometry chambers and, submitted to progressive hypoxia for the determination of critical PO2. In a subsequent experiment, the fish received an intraperitoneal injection of naphthalene and were kept for 96 hours under normoxia. Successively, fish were challenged with acute hypoxia (PO2<PO2 crit) during 6 hours. We observed that the PO2 crit was not affected by naphthalene injection. Moreover, hematological parameters were modulated only in response to hypoxia. Fish with naphthalene injection plus hypoxia exposure presented altered activity of the GST and CAT enzymes. Exposure to naphthalene also resulted in DNA damages, which was not influenced by hypoxia. Hypoxia accentuated the hepatic lesions caused by naphthalene, as well as it also impaired the transcription of tp53 in naphtalene injected fish, demonstrating the risks of contaminating aquatic environments, especially environments where hypoxic conditions are common and occur on a daily or on seasonal basis, as in the Amazon basin.