Escherichia coli is a key indicator of faecal contamination and a reservoir of antimicrobial resistance genes (ARGs) in aquatic environments. Monitoring its environmental prevalence and resistance patterns in water sources is essential for public health and environmental safety. In this study, a total of 300 water samples were collected from diverse sources. E. coli were isolated from 32% samples, with the highest prevalence in river water (72.72%) and the lowest in tap water (10%). All isolates were positive for the yaiO gene. Complete (100%) resistance was observed against ampicillin-sulbactam and polymyxin-B, while high resistance was displayed by isolates against Tigecycline (94.6%), ciprofloxacin (94.4%), colistin (94.4%), and amikacin (94.4%). Comparatively, higher sensitivity was noted to azithromycin (46%) and meropenem (41.1%). PCR analysis detected tetA/tetB genes in 37.5% and dfrA in 20.83% of isolates, with significant variation in gene distribution across water sources. The study revealed a substantial prevalence of multidrug-resistant E. coli in surface and groundwater sources, posing a potential risk to human and animal health. The presence of ARGs highlights the aquatic environment as a reservoir for resistance determinants, underscoring the need for regular water quality surveillance, improved sanitation, and prudent antibiotic stewardship within a One Health framework.