- Enzyme–substrate microbial water tests, originally developed for efficiency gains in laboratory settings, are potentially useful for on-site analysis in remote settings. This is especially relevant in developing countries where water quality is a pressing concern and qualified laboratories are rare. We investigated one such method, Colisure, first for sensitivity to incubation temperatures in order to explore alternative incubation techniques appropriate for remote areas, and then in a remote community of Zambia for detection of total coliforms and Escherichia coli in drinking-water samples. We sampled and analyzed 352 water samples from source, transport containers and point-of-use from 164 random households. Both internal validity (96–100%) and laboratory trials (zero false negatives or positives at incubation between 30 and 40 °C) established reliability under field conditions. We therefore recommend the use of this and other enzyme-based methods for remote applications. We also found that most water samples from wells accessing groundwater were free of E. coli whereas most samples from surface sources were fecally contaminated. We further found very low awareness among the population of the high levels of recontamination in household storage containers, suggesting the need for monitoring and treatment beyond the water source itself.