One major health issue is the microbial and chemical contamination of natural freshwater, particularly in Latin American countries, such as Ecuador, where it is still lacking wastewater treatment plants. This study analyzed the water quality in twelve rivers of Ecuador (Coastal, Andean, and Amazonian regions). All rivers showed levels of E. coli and total coliforms above the maximum limit according to International and Ecuadorian legislations. The most polluted rivers were Zamora, Esmeraldas and Machángara. Also, E. coli pathotypes were found in six rivers. Several physicochemical and metal parameters were detected in high levels, such as CODTOTAL (in eight rivers), TSS (in six rivers), TS (in two rivers), Al (in nine rivers), Zn (in eight rivers), Pb (in three rivers), Cu (in three rivers), Fe (in two rivers), and Mn (in Machángara River). Our results agree with other studies in Latin America (such as Colombia, Brazil, and Peru) reporting similar contamination in water resources used for agriculture, livestock, and human consumption. Overall, Guayas, Guayllabamba, and Machángara Rivers showed the highest levels of physicochemical parameters (such as CODTOTAL and TSS) and metal concentrations (such as copper, zinc, aluminum, iron, and manganese). Further studies should evaluate contamination sources and public health impact.