The global growth of antimicrobial resistance (AMR) raises significant challenges to public health, necessitating comprehensive surveillance and intervention strategies. This study investigates the prevalence and resistance profiles of Escherichia coli isolated from three different wastewater treatment plants (WWTPs) in Romania during the warm season of 2023. Through systematic sampling and subsequent microbiological and molecular analyses, we identified a 50% prevalence of E. coli in wastewater samples, with a concerning 83.34% of isolates displaying resistance to multiple antibiotics. The resistance was notably high against ampicillin, ticarcillin/clavulanic acid, and cefalotin, with observed multidrug resistance suggesting a robust presence of antibiotic-resistant bacteria (ARB) within treated effluents. Molecular characterization confirmed the presence of multiple antibiotic resistance genes (ARGs), including β-lactamase producers and markers for tetracycline and sulphonamide resistance. These findings underscore the critical role of WWTPs as both reservoirs and potential dissemination points for ARB and ARGs, highlighting the need for integrated surveillance systems and enhanced wastewater treatment protocols to mitigate the spread of AMR. This study emphasizes the necessity of bridging clinical and environmental monitoring to develop effective public health strategies against the threat of antibiotic resistance.