Microbial communities and their associated enzyme activities affect the amount and chemical quality of carbon (C) in soils. Increasing nitrogen (N) deposition, particularly in N-rich tropical forests, is likely to change the composition and behavior of microbial communities and feed back on ecosystem structure and function. This study presents a novel assessment of mechanistic links between microbial responses to N deposition and shifts in soil organic matter (SOM) quality and quantity. We used phospholipid fatty acid (PLFA) analysis and microbial enzyme assays in soils to assess microbial community responses to long-term N additions in two distinct tropical rain forests. We used soil density fractionation and 13C nuclear magnetic resonance (NMR) spectroscopy to measure related changes in SOM pool sizes and chemical quality. Microbial biomass increased in response to N fertilization in both tropical forests and corresponded to declines in pools of low-density SOM. The chemical quality of this soil C pool reflected ecosystem-specific changes in microbial community composition. In the lower-elevation forest, there was an increase in gram-negative bacteria PLFA biomass, and there were significant losses of labile C chemical groups (O-alkyls). In contrast, the upper-elevation tropical forest had an increase in fungal PLFAs with N additions and declines in C groups associated with increased soil C storage (alkyls). The dynamics of microbial enzymatic activities with N addition provided a functional link between changes in microbial community structure and SOM chemistry. Ecosystem-specific changes in microbial community composition are likely to have far-reaching effects on soil carbon storage and cycling. This study indicates that microbial communities in N-rich tropical forests can be sensitive to added N, but we can expect significant variability in how ecosystem structure and function respond to N deposition among tropical forest types.