Artisanal and small-scale gold mining (ASGM) is a major source of mercury (Hg) contamination in Amazonian ecosystems, due to remobilization of geogenic Hg from mined soil, and anthropogenic Hg used for gold amalgamation. To assess these two relative contributions, Hg stable isotope composition together with geochemical variables were determined in sediment samples collected in the early 2000s across the main watersheds of French Guiana. Mercury in sediment was found associated with organic matter and Fe/Al oxides. Total Hg concentrations were correlated (p < 0.001) to both mass-dependent (δ202Hg) and odd-mass-independent fractionation (Δ199Hg) indicating that the Hg isotopic composition of river sediments reflects the extent of contamination, with two isotopic end-members for pristine or geogenic (δ202Hg = −2.53 ± 0.23‰, Δ199Hg = −0.62 ± 0.10‰) and for ASGM contaminated (δ202Hg = −0.42 ± 0.20‰, Δ199Hg = −0.01 ± 0.07‰) sediments. A binary mixing model showed that the contribution of anthropogenic Hg in river sediments varied widely (ca. 0 to 100%) and reflected the geographical and historical monitoring of ASGM activities in French Guiana. These results highlight that Hg isotopic measurements in archived sediments allow retrospective assessment of Hg pollution in ASGM-impacted regions and evaluation of its temporal evolution.