We report on the effect of single and mixed infections with two gut symbionts, trypanosomatids and the intracellular fungus Coccidiascus legeri, on the life history of their host, Drosophila melanogaster. We also provide the first report on the prevalence of C. legeri in natural populations of Drosophila. Prevalence overall was low (3.4%), and differed with host species, but persisted from the first to the second year of our survey. We documented delayed pupation in flies exposed to trypanosomatids, but larvae exposed to the fungus eclosed more quickly than controls. Larvae exposed to mixed infections pupated more slowly, but eclosed more quickly than controls.