Aims: We aimed to develop an assay for sensitive detection and quantification of enterotoxigenic Escherichia coli (ETEC) in different types of water samples. Methods and Results: Real-time polymerase chain reaction (PCR) assays with primers against ETEC enterotoxin genes estA (STh) estB (STp) and eltB (LT) were designed and the detection levels were determined to be three bacteria per PCR reaction. Gene copy numbers were estimated to be four (LT), two (STh) and one (STp) per bacteria. Twenty-six household and 13 environmental water samples from Bangladesh were filtered through 0.22-mu m filters; DNA was extracted from the filters and analysed by real-time PCR. The results were compared with toxin GM1-enzyme-linked immunosorbent assay (ELISA), in which colonies were tested for toxin production after cultivation of the filters. Out of the 39 samples tested, 18 household and 8 environmental samples were positive for ETEC in real-time PCR, but only 6 positive samples were found with GM1-ELISA. Conclusions: The method allows for highly sensitive detection and quantification of ETEC based on detection of toxin DNA in water samples. Significance and Impact of the Study: The method facilitates detection and identification of ETEC in water and allows comparison between water contamination and incidence of ETEC diarrhoea in endemic areas.