Microbes are important in stream ecosystem processes and ubiquitous in stream environments, but limitations of study techniques have left most of these microbial communities poorly described. In stream ecosystems, fungal and bacterial communities play critical roles in leaf decomposition and release energy and nutrients to higher trophic levels of the food web. Our research examined microbial communities in Fossil Creek, Arizona, USA, to elucidate effects of litter quality and abiotic habitat characteristics on early microbial colonizers of leaves. High- and low-quality leaf litter was placed in the creek at 5 study sites with heterogeneous environmental conditions (including differing stream morphology, water flow, water chemistry, and travertine deposition). Microbial assemblages that colonized the decomposing leaves were characterized using terminal-restriction fragment length polymorphism analysis and clone library comparisons. Our study revealed differences in microbial community structure along environmental gradients and, to some extent, between high- and low-quality litter in Fossil Creek. Leaf decomposition rates were strongly influenced by both litter quality and abiotic site characteristics, but microbial communities were more strongly influenced by site than by litter quality. Bacterial and fungal communities differed with incubation times: bacterial diversity increased between 2-d and 8- to 9-d incubations, whereas fungal diversity decreased. Fungal community diversity was negatively correlated with decomposition rates after incubation in the creek for 2 d when the community still included nonaquatic fungi, but this relationship did not exist after longer incubation. Bacterial community diversity was not related to litter quality or decomposition rates.