Fecal contamination of water sources represents a significant public health risk, particularly in regions with limited sanitation infrastructure. This study characterized diarrheagenic Escherichia coli (DEC) pathotypes, antimicrobial resistance, and clonal relatedness among E. coli isolates recovered from surface and groundwater sources in the Upper Choluteca River Basin, Honduras. In total, 131 isolates were screened by PCR for nine marker genes of six diarrheagenic pathotypes and tested for susceptibility to seven antimicrobial agents; DEC isolates were typed by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Overall, 65.6% (86/131) of the isolates carried at least one DEC marker; enterotoxigenic E. coli (ETEC) predominated (46.6%), and 13.7% showed heteropathotype profiles, mainly typical enteropathogenic E. coli (tEPEC)/ETEC. Multidrug resistance was identified in 71.0% of the isolates, with the highest non-susceptibility frequencies for cefotaxime (95.4%) and ciprofloxacin (93.1%); imipenem non-susceptibility (51.9%) was mostly intermediate (resistance, 8.4%). The st gene was significantly associated with non-susceptibility to cefoxitin, imipenem, and ceftazidime. ERIC-PCR revealed extensive clonal diversity without a dominant lineage, suggesting recurrent fecal inputs from multiple sources. These findings show that water sources in the basin, including groundwater, are reservoirs of DEC and multidrug-resistant E. coli, highlighting the need for stronger water-quality surveillance within a One Health framework.