The area of Ponce Enríquez, Southern Ecuador, is one of the most important artisanal and small scale gold mining (ASGM) areas in the country, where informal mining operations can be found using amalgamation technique, even though it has been prohibited since 2010. Considering the potential impact of these mining operations, the present research focused on heavy metal pollutants in subsurface sediments at 214 sampling locations from several detailed sampling campaigns in the rivers Fermín, Fermín Norte, Guanache, Villa, and Siete. Four parameters were calculated to evaluate the geochemical composition of the river sediment and heavy metal concentrations: The enrichment factor (EF), the geo-accumulation index (Igeo), the contamination factor (Cf) and the pollution load index (PLI). The analyzed elements were Cu, Pb, Zn, Ni, As, Cd, Sb, Sr, and Hg. Due to the lack of standards for pollution levels in sediments in Ecuador, Canadian guidelines were used as interim sediment quality guidelines (ISQG) in this research for comparison with the obtained results in subsurface sediments. The multi-index geochemical analysis indicated elevated heavy metal concentrations in all subsurface sediments in the studied river ecosystems.