The present study was undertaken to assess the drinking water quality of well water with respect to Escherichia coli contamination from different sources. A total of 138 drinking water samples contaminated with coliforms above 10 MPN Index per 100 mL formed the study material. A cross sectional survey was conducted for the sanitary evaluation of drinking water along with inspection of household wells and surroundings. A retrospective investigation was carried out to assess the contamination of E. coli-positive well water samples. The associated sources for E. coli contamination like, 93 human stool samples, 84 animal faecal samples, 25 bird cloacal swabs, 138 soil and 27 drainage water samples were also collected from the premises of the respective households in order to identify the source contaminating the well water. All the samples were subjected to isolation and identification of E. coli by conventional culture technique followed by molecular confirmation using polymerase chain reaction (PCR). Statistical analysis using Pearson’s chi square test revealed highly significant association between the occurrence of E. coli in drinking water and sources viz., human stool, animal faeces and soil samples (p ≤ 0.01). The unassociated sources of E. coli contamination in drinking water exhibited a highly significant association with the increasing distance of well from the septic tank as well as with the presence of linings in wells and animals in the households. Hence, the study conducted for the identification of sources of E. coli contamination of drinking water signifies the importance of revised construction rules to protect the wells from contaminating sources and the need of regular and effective water treatment protocols.