Owing that Oreochromis niloticus is resistant to different aquatic environments, four contaminated sites were selected: decentralized engineered ecosystem (DEE) and conventional wastewater treatment plants (WWTP); urban lagoon; and gasolinecontaminated marshland. Endocrine disruption was assessed by alkali-labile phosphate (ALP) quantification, genotoxicity by micronuclei frequency, and morphological changes by hepatosomatic and gonadosomatic indexes. The ALP baseline of non-exposed O. niloticus males was low, compared with other fish species in the literature. Endocrine disruption was observed in fish exposed to raw wastewater of WWTP and DEE, discharge point of channeled river in the urban lagoon, and water from gasoline-contaminated marshland. After treatment in the DEE, the endocrine disruption effect was removed. The micronuclei frequency corroborated with the ALP results in most cases and proved to be a useful tool to assess genotoxicity in fish. The exposure time of 15 days was not enough to provoke morphological effects in most samples. However, in all gasoline-contaminated samples, the fishes showed an increase in the hepatosomatic index followed by a decrease in the gonadosomatic index. The tested biomarkers showed feasibility, sensibility, reproducibility, and were complementary in the assessment of chronic ecotoxicity; therefore, we recommend them to compose a suitable protocol for aquatic monitoring programs.