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Escherichia coli, is intimately associated with both human health and water sanitation. E. coli isolates from water can either be (i) host associated commensals, indicating recent faecal contamination; (ii) diarrheal pathogens or (iii) extra-intestinal pathogens that pose a direct health risk; or (iv) free-living. In this study we genetically characterised 28 E. coli isolates obtained from treated drinking water in south eastern Australia to ascertain their likely source. We used full genome sequencing to assign the isolates to their phylogenetic group and multi-locus sequence type. The isolates were also screened in silico for several virulence genes and genes involved in acquired antibiotic resistance. The genetic characteristics of the isolates indicated that four isolates were likely human pathogens. However, these isolates were not detected in sufficient numbers to present a health risk to the public. An additional isolate was a human associated strain. Nine isolates were water associated free-living strains that were unlikely to pose a health risk. Only 14% of the isolates belonged to the host associated phylogenetic group (B2) and only a single isolate had any antibiotic resistance genes. This suggests that the primary source of the drinking water E. coli isolates may not have been recent human faecal contamination.