Unsafe water sources contaminated with fecal bacteria and antibiotic-resistant pathogens represent a critical public health challenge in rapidly urbanizing regions. This study investigates the occurrence, distribution, and antimicrobial resistance (AMR) patterns of Escherichia coli across interconnected water sources in Guwahati, India, including river water, wells, municipal supply, and groundwater. A total of 87 samples were analyzed using membrane filtration, biochemical identification, and automated confirmation, followed by antibiotic susceptibility testing and phenotypic detection of β-lactamase production. Total coliforms were detected in 64.4% of samples, while E. coli was confirmed in 51.7%. According to World Health Organization risk thresholds, 93.3% of river water samples and 50% of groundwater samples fell within the very-high-risk category (>1000 CFU/100 mL), indicating widespread fecal contamination in sources used for domestic purposes. Antibiotic-resistant E. coli was identified across all water types, with 14.1% of isolates exhibiting multidrug resistance (MAR index > 0.2). Notably, 20.6% of isolates produced β-lactamases, including one extended-spectrum β-lactamase (ESBL) producer, while no carbapenemase-producing strains were detected. The co-occurrence of multidrug-resistant E. coli across surface and groundwater systems is consistent with the interconnected nature of urban water contamination and suggests the potential for widespread exposure through drinking water pathways, though direct transfer between water systems was not experimentally confirmed in this study. These findings underscore the urgent need for improved wastewater management, protection of groundwater resources, and strengthened antibiotic stewardship within a One Health framework to mitigate the environmental dissemination of AMR.