The water sources in developing countries are usually contaminated with the members of enterobacteriaceae leading to gastrointestinal infections. There has been an increasing consumption of antibiotics during last decade in India. The development of multiple antibiotic resistance and heavy metal tolerance among waterborne bacteria has been coexisting traits. Keeping above features in view, 51 waterborne isolates of Escherichia coli O157:H7 have been characterized in the present study. The disk diffusion test for antibiotic sensitivity assay revealed that all the isolates were resistant to at least one or more antibiotics. Cephalosporins were least effective, while fluoroquinolones and aminoglycosides were most effective antibiotics. The isolates shared resistance pattern similarities regardless of their sources. The isolates were subjected to test maximum tolerance concentration of heavy metals such as arsenic, lead, chromium and mercury. About 98% isolates tolerated arsenic at 250 μg ml−1 or various above concentrations. Mercury >25 μg ml−1 was not tolerated by more than 90% isolates. In dug well, a commonly used water source in rural India, the isolates exhibited multiple antibiotic resistance index of 0.68 indicating it as high risk contaminating source. PCR–RFLP of 16S rDNA distinguished isolates into four groups with the smallest group represented by 3 isolates. Three isolates belonged to different sources but shared all the features of biochemical, serological, multiple antibiotic resistance and heavy metal tolerance test. 16S rDNA sequence analysis showed that the three selected isolates were closely related to each other. Based on the microbial and molecular findings, the study recommends to follow social cognitive theory to alter the behavior of the local communities for sustainable use of water resources.