Mercury (Hg) poses a public health burden in the Amazon and worldwide. Although usually attributed to Hg used in artisanal and small-scale gold mining (ASGM), the primary source of elevated Hg in Amazonian aquatic ecosystems is contested since there have not been tools to differentiate between Hg from ASGM and Hg from other sources such as increased soil erosion associated with landcover and land-use change. To directly assess Hg contamination from ASGM, stable Hg isotope analyses were applied to sediment cores, surface sediments, and soils from two aquatic ecosystems in Amapa, Brazil, one downstream of ASGM activities and one isolated from ASGM. Downstream of the ASGM sites, the Hg isotope data is consistent with elevated Hg coming dominantly from increased erosion of soils and not from Hg used during gold extraction. Although these two sources represent different pathways of contamination to downstream ecosystems, ASGM may contribute to both land-cover and land-use change and local contamination of soils. Accordingly, these findings demonstrate that in some regions of the Amazon effective Hg mitigation strategies need to address land-use practices in addition to ASGM.