Water can act as a vector for several microbes with significant pathogenic potential for both humans and animals. Waterborne infections are a critical public health concern as they cause more than 3.4 million deaths annually. Total and thermotolerant coliforms and intestinal enterococci have traditionally been used to assess the quality and suitability of drinking water. The aim of this study was to evaluate the microbiological quality of groundwater from six sub-basins located in the upper Choluteca River basin in Honduras and to determine the E. coli phylogroups isolated in these samples. Our findings show high rates of fecal contamination, which suggests that the groundwater in the basin is unsafe for human consumption. Phylogroups B1 and D were the most frequent among 99 E. coli isolates, while C and F were the least frequent phylogroups. Measures must be taken to raise awareness about sanitation and good practices for the management of household waste as well as the waste generated by agro-industrial activity and livestock.