Niche differentiation among species is a key mechanism by which biodiversity may be linked to ecosystem function. We tested a set of widely invoked hypotheses about the extent of niche differentiation in one of the most diverse communities on Earth, decomposer microorganisms, by measuring their response to changes in three abundant litter resources: lignin, cellulose, and nitrogen (N). To do this, we used the model system Arabidopsis thaliana to manipulate lignin, cellulose, and N availability and then used high-throughput sequencing to measure the response of microbial communities during decay. Resequencing the decomposer communities after incubation of decomposed litter with pure substrates showed that groups of species had unique substrate use profiles, such that species organized into functional “guilds” of decomposers that were associated with individual litter chemicals. Low concentrations of lignin, cellulose, or N in the litter caused unique shifts in decomposer community composition after 1 yr of decay. Low cellulose plants had low levels of fungi in all decomposer guilds, low lignin plants had high levels of fungi in all decomposer guilds, and low N plants had low levels of fungi in decomposer guilds associated with sucrose and lignin. The relative abundance of decomposer guilds correlated with the total loss of individual litter chemicals during litter decay in the field. In addition, N fertilization shifted decomposer communities during both the early and later stages of decay to those dominated by decomposers in the cellulose guild. Our results contrast the assumption that major carbon (C) and N degradation mechanisms are uniform across whole decomposer communities and instead suggest that decomposition arises from complementarity among groups of metabolically distinct taxa.