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Sachet water is one of the primary sources of drinking water in rapidly growing countries. A study to assess the microbiological quality of sachet water in 21 different brands was conducted in Ghana. Culturable total coliform was positive in 87% of the samples collected, where Escherichia coli colonies were absent. The analysis of quantitative polymerase chain reaction results indicated the presence of E. coli genes in 44.6% of the samples, with the highest concentration up to 3,166 CCE/100 ml. Microbial source tracking analyses showed that the source of E. coli genes did not originate from sewage contamination because the human-associated HF183 marker was not detected. Of the 175 samples tested, 71% did not mention any water treatment before filling the packages. These results suggest non-human sources of contamination, such as biofilm formation in the pipelines used to fill these packages due to poor disinfection. Our study shows an urgent need for increased regulation and standardized manufacturing of sachet water to ensure safe drinking water.