Due to its selectivity and specificity, the Colilert methodology allows one to detect the sanitation indicators of water quality, total Coliforms and Escherichia coli. The aim of this study was to test the Colilert methodology applied to a natural water matrix. With a variation coefficient (CV) less than 6% and 7% for total Coliforms and E. coli respectively, this methodology has proven its repeatability. For both indicators, the middle accuracy was CV < 6% and percentages of recuperation were between 80–120%. The results proved that this method is precise, repeatable and accurate, according to the acceptance criteria entered in the Colombian Technique Guide GTC 84 from 2003; the Standard methods for the examination of water and the International Rule ISO/9308–2:2012. Thus, the method is suitable for detecting and quantifying total Coliforms and E. coli, while providing truthful and timely results for customers.