Evaluation of habitat preference and ecological interactions of pathogenic environmental bacteria may be key to understanding the emergence of many diseases. Buruli ulcer is an emerging disease caused by the bacterium Mycobacterium ulcerans. The disease occurs in 30 countries worldwide, but transmission pathways and pathogen habitat preferences are poorly understood. In 2006, a survey of waterbodies across several regions of Ghana revealed correlations between waterbody phosphorous and dissolved oxygen with Buruli ulcer cases, but not with M. ulcerans. However, diatom assemblages on macrophytes predicted the likelihood of macrophytes to harbor M. ulcerans, suggesting habitat specificity. To determine if M. ulcerans would preferentially colonize biofilms grown under different nutrient regimes, we compared M. ulcerans colonization and algal biomass between nutrient-diffusing substrata supplemented with N, P, N+P, or glycerol in a southern Ghana wetland. Algal biomass was significantly higher on N and N+P substrata compared to controls (p<0.001), suggesting algae was N-limited. However, M. ulcerans colonization was significantly lower on N+P substrata than controls (p=0.02). These data indicate that M. ulcerans may not colonize thick algal biofilms as readily as other habitats and suggest the importance of identifying the preferred microhabitat of this pathogen in understanding disease transmission.