ABSTRACT Environmental dissemination of multidrug resistance (MDR) has emerged as a global concern worldwide, particularly in developing countries like Bangladesh where the waste disposal system is inadequate. The present investigation was intended to comprehend the drug resistance and virulence pattern of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) in surface water samples (n = 16) of Bangladesh, which are adjoining poultry farms, agricultural fields, pharmaceutical companies, and textile industries. A total of 61 E. coli with ESBL production were identified from 16 water samples based on phenotypic and genotypic approaches. MDR was observed in 96% (n = 59) of the isolates with the multiple antibiotic resistance (MAR) index in the range of 0.3–0.9. The most prevalent MDR phenotype was non-susceptible to Ampicillin–Azithromycin–Nalidixic acid, with the highest percentage in the isolates from samples near agricultural fields followed by poultry farms. The frequencies of three ESBL resistance genes were detected as blaTEM (63%), blaCTX-M-13 (60%), and blaSHV (14%). Approximately 11% of the E. coli isolates were revealed as virulent gene positive, with the predominant one (eagg) specific for Enteroaggregative E. coli (EAEC). This study implies that the aquatic environment could be a potent means of exposure and transmission of bacterial antibiotic resistance and their genetic determinants in Bangladesh.