Environmental isolates of the fungus Rhizopus have been shown to harbor a bacterial endosymbiont (Burkholderia) that produces rhixozin, a plant mycotoxin. We sought to define the role of rhizoxin production by endosymbionts in the pathogenesis of mucormycosis. Endosymbiotic bacteria were identified by polymerase chain reaction in 15 (54%) of 28 clinical isolates of Zygomycetes, with 33% of the bacterial strains showing 87% identity to Burkholderia 16S rDNA. The presence of rhizoxin in myclial extracts from fungi harboring bacteria was confirmed by high-performance liquid chromatography analysis. However, fungal strains with or without endosymbionts did not differ in their ability to cause endothelial cell injury in vitro, nor did antibiotic-mediated eradication of endosymbionts and rhizoxin production decrease the virulence of fungal strains in mice or flies. In summary, although bacterial endosymbiosis is widely detected in clinical isolates of Zygomycetes, including Rhizopus oryzae strains, we found no evidence that bacterial endosymbionts and rhizoxin contribute to the pathogenesis of mucormycosis in the models studied.