Organic carbon (C) and nitrogen (N) are essential for heterotrophic soil microorganisms, and their bioavailability strongly influences ecosystem C and N cycling. We show here that the natural 15N abundance of the soil microbial biomass is affected by both the availability of C and N and ecosystem N processing. Microbial 15N enrichment correlated negatively with the C : N ratio of the soil soluble fraction and positively with net N mineralization for ecosystems spanning semiarid, temperate and tropical climates, grassland and forests, and over four million years of ecosystem development. In addition, during soil incubation, large increases in microbial 15N enrichment corresponded to high net N mineralization rates. These results support the idea that the N isotope composition of an organism is determined by the balance between N assimilation and dissimilation. Thus, 15N enrichment of the soil microbial biomass integrates the effects of C and N availability on microbial metabolism and ecosystem processes.