Abstract Background Leptospira are shed into the environment via urine of infected animals. Rivers are thought to be an important risk factor for transmission to humans, though much is unknown about the types of environment or characteristics that favor survival. To address this, we screened for Leptospira DNA in two rivers in rural Ecuador where Leptospirosis is endemic. Results We collected 112 longitudinal samples and recorded pH, temperature, river depth, precipitation, and dissolved oxygen. We also performed a series of three experiments designed to provide insight into Leptospira presence in the soil. In the first soil experiment, we characterized prevalence and co-occurrence of Leptospira with other bacterial taxa in the soil at dispersed sites along the rivers (n = 64). In the second soil experiment, we collected 24 river samples and 48 soil samples at three points along eight transects to compare the likelihood of finding Leptospira in the river and on the shore at different distances from the river. In a third experiment, we tested whether Leptospira presence is associated with soil moisture by collecting 25 soil samples from two different sites. In our river experiment, we found pathogenic Leptospira in only 4 (3.7%) of samples. In contrast, pathogenic Leptospira species were found in 22% of shore soil at dispersed sites, 16.7% of soil samples (compared to 4.2% of river samples) in the transects, and 40% of soil samples to test for associations with soil moisture. Conclusions Our data are limited to two sites in a highly endemic area, but the scarcity of Leptospira DNA in the river is not consistent with the widespread contention of the importance of river water for leptospirosis transmission. While Leptospira may be shed directly into the river, onto the shores, or washed into the river from more remote sites, massive dilution and limited persistence in rivers may reduce the environmental load and therefore, the epidemiological significance of such sources. It is also possible that transmission may occur more frequently on shores where people are liable to be barefoot. Molecular studies that further explore the role of rivers and water bodies in the epidemiology of leptospirosis are needed.