Abstract Mercury (Hg) is a toxic metal subject to several international regulations. The European Water Framework Directive (WFD) established in 2008 an Environmental Quality Standard for biota (EQSbiota) at 0.02 µg.g−1 fresh weight. This standard is not always adapted, such as in French Guiana subjected to high natural background Hg levels and intensive illegal gold mining. Therefore, this study focuses on how to apply the WFD for the definition of good chemical status (i.e., EQSbiota) in a context of strong and generalized natural and anthropic Hg contamination. Based on Hg concentrations measured in 6208 fish over >200 sites between 2004 and 2015, we first aimed at discriminating the natural or anthropogenic influences at each site. Then, as WFD recommends considering only high trophic level fish species as bioindicator species, we selected carnivorous/piscivorous fish species able to significantly accumulate Hg and discriminate reference sites from gold mining polluted sites. Total Hg concentrations measured in fish muscle were mostly above the EQSbiota (100% for creeks and 84% for rivers), confirming the unsuitability of the direct application of this standard in French Guiana. Among the studied sites, few potential reference sites were identified: eight sites spread over six different watersheds for creeks, and only two areas (group of sites) both on the Oyapock watershed for rivers. Several relevant bioindicators fish species are proposed: ten species (over 35 species tested) belonging to seven genera on creeks (Moenkhausia oligolepis, Gymnotus carapo, Sternopygus macrurus, Jupiaba [abramoides + keithi], Pimelodella [cristata + geryi + macturki], Copella carsevennensis, Pyrrhulina filamentosa.), and four species (over 21 species tested) belonging to three genera on rivers (Acestrorhyncus [micropelis + falcatus], Hoplias aimara, Ageneiosus inermis). In order to facilitate field sampling, difficult in such remote hydrosystems, and to improve results interpretation, we tested the possibility to group some of these species. Our results indicate that only Jupiaba, Moenkhausia, Pimelodella and Pyrrhulina on creeks could be grouped; and the three bioindicators species proposed on rivers could be pooled. Finally, this work proposes in situ-based reference Hg concentrations for selected bioindicator fish species from French Guiana as an alternative to detect Hg-impacted sites and help the application of the WFD in tropical systems.