Presence of Vibrio cholerae serogroups O1 and O139 in the waters of the rural area of Matlab, Bangladesh, was investigated with quantitative measurements performed with a portable flow cytometer. The relevance of this work relates to the testing of a field-adapted measurement protocol that might prove useful for cholera epidemic surveillance and for validation of mathematical models. Water samples were collected from different water bodies that constitute the hydrological system of the region, a well-known endemic area for cholera. Water was retrieved from ponds, river waters, and irrigation canals during an inter-epidemic time period. Each sample was filtered and analysed with a flow cytometer for a fast determination of V. cholerae cells contained in those environments. More specifically, samples were treated with O1- and O139-specific antibodies, which allowed precise flow-cytometry-based concentration measurements. Both serogroups were present in the environmental waters with a consistent dominance of V. cholerae O1. These results extend earlier studies where V. cholerae O1 and O139 were mostly detected during times of cholera epidemics using standard culturing techniques. Furthermore, our results confirm that an important fraction of the ponds’ host populations of V. cholerae are able to self-sustain even when cholera cases are scarce. Those contaminated ponds may constitute a natural reservoir for cholera endemicity in the Matlab region. Correlations of V. cholerae concentrations with environmental factors and the spatial distribution of V. cholerae populations are also discussed.