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Knowledge regarding the potential impacts of crude oil on endocrine signalling in freshwater aquatic vertebrates is limited. In this study, the expression of selected genes as biomarkers for altered endocrine signalling were studied in African clawed frog, Xenopus laevis tadpoles, and juvenile Mozambique tilapia, Oreochromis mossambicus exposed to weathered bunker and un-weathered refinery crude oil water accommodated fractions (WAFs). In addition, the expression of the aforementioned genes was quantified in X. laevis tadpoles exposed to surface water collected from the proximity of an underground oil bunker. The (anti)estrogenicity and (anti)androgenicity of crude oil, crude oil WAFs and surface water were furthermore evaluated using recombinant yeast. Thyroid hormone receptor beta expression was significantly downregulated in X. laevis in response to both oil WAF types, whereas, a further thyroid linked gene, type 2 deiodinase was upregulated in O. mossambicus exposed to a high concentration of bunker oil WAF. In addition, both WAFs altered the expression of the adipogenesis-linked peroxisome proliferator-activated receptor gamma in X. laevis. The crude oil and WAFs exhibited anti-estrogenic and anti-androgenic activity in vitro. However, O. mossambicus androgen receptor 2 was the only gene, representing the reproductive system, significantly affected by WAF exposure. Estrogenicity, anti-estrogenicity and anti-androgenicity were detected in surface water samples; however, no significant changes were observed in the expression of any of the genes evaluated in X. laevis exposed to surface water. The responses varied among the two model organisms used, as well as among the two types of crude oil. Nonetheless, our data provide evidence that crude oil pollution may lead to adverse health effects in freshwater fish and amphibians due to altered endocrine signalling. This article is protected by copyright. All rights reserved