Buruli ulcer is a neglected infectious disease often found in developing nations. The natural reservoir and mode of transmission of the bacterial pathogen, Mycobacterium ulcerans, remain unknown but are thought to be associated with ecologically disturbed aquatic habitats. To better understand the ecology of this disease a large-scale bioassessment of 98 waterbodies of Ghana, Africa, was undertaken from 2005-2007 to determine, (1) the distribution of M. ulcerans in the aquatic environment, and (2) how landscape varied with M. ulcerans distribution and human disease outbreaks. Results show that land use is significantly related to BU cases at large spatial scales, while water quality and macrophyte type are associated with disease at local scales. Interestingly, the distribution of human disease presence and the pathogen did not overlap, corresponding to different environmental factors driving pathogen and disease spatial differentiation among waterbodies. These results suggest that other variables such as season-driven temporal variation, or specific human behaviors are involved with disease transmission. Alternatively, the commonly held belief that transmission occurs within aquatic habitats may be erroneous. Nonetheless, these results demonstrate the importance of understanding the ecology of infectious diseases for testing anecdotal assumptions of pathogen ecology common in epidemiology.