Since the middle of the 1500s, mining has been active in central Mexico. Total estimates for low-grade piles and mine tailing materials in the Guanajuato mining district (GMD) are in the range of 150 million tons, covering an area of 15 to 20 km2. GMD is located in the Guanajuato River sub-basin (GRB), which is part of one of the largest basins in Mexico (Lerma-Santiago). Previous studies on the GRB found unusually high concentrations of heavy metals in mining tailings and sediments. Geochemical and statistical methods were used here to determine the sediment's origin, background values, degree of contamination, and toxicity through different contamination indices. This analysis shows that Cu, Co, As, Sb, and Hg are higher than they are in the upper continental crust (UCC) overbank sediments without human and mining influence, because of the ore deposits and rock weathering in GRB. Geochemistry results in stream sediments show anomalies, where Hg, Cu, Zn, As, and Pb are higher than UCC because those heavy metals and trace elements (HMT) have been influenced by human activities and mineral recovery (smelting, amalgamation, cyanidation). The distribution of high concentrations of HMTs and contamination indices occur in the main channel of the Guanajuato River and downstream of the city of Guanajuato. Statistical analyses (cluster and principal component analysis) reveal relationships between Cr, Ni, Cu, and Pb, which are primarily of natural origin, related to rocks of the upper basin. The middle and lower basins are distinctive in their associations between As, Sb, Zn, Pb, and Hg. Additionally, it is recognized that the origins of Pb, Zn, and Hg are geogenic and anthropogenic. This study demonstrates how crucial it is to understand the geochemistry of various HMT sources, with both natural and anthropogenic contributions (stream sediments and rocks), in order to calculate a more realistic background in a basin with both natural anomalies and anthropogenic contamination. The basin is a regional aquifer recharge area, so the new geochemical data are important for improving basin environmental management.