The effects of atmospheric nitrogen (N) deposition on organic matter decomposition vary with the biochemical characteristics of plant litter. At the ecosystem-scale, net effects are difficult to predict because various soil organic matter (SOM) fractions may respond differentially. We investigated the relationship between SOM chemistry and microbial activity in three northern deciduous forest ecosystems that have been subjected to experimental N addition for 2 years. Extractable dissolved organic carbon (DOC), DOC aromaticity, C : N ratio, and functional group distribution, measured by Fourier transform infrared spectra (FTIR), were analyzed for litter and SOM. The largest biochemical changes were found in the sugar maple–basswood (SMBW) and black oak–white oak (BOWO) ecosystems. SMBW litter from the N addition treatment had less aromaticity, higher C : N ratios, and lower saturated carbon, lower carbonyl carbon, and higher carboxylates than controls; BOWO litter showed opposite trends, except for carbonyl and carboxylate contents. Litter from the sugar maple–red oak (SMRO) ecosystem had a lower C : N ratio, but no change in DOC aromaticity. For SOM, the C : N ratio increased with N addition in SMBW and SMRO ecosystems, but decreased in BOWO; N addition did not affect the aromaticity of DOC extracted from mineral soil. All ecosystems showed increases in extractable DOC from both litter and soil in response to N treatment. The biochemical changes are consistent with the divergent microbial responses observed in these systems. Extracellular oxidative enzyme activity has declined in the BOWO and SMRO ecosystems while activity in the SMBW ecosystem, particularly in the litter horizon, has increased. In all systems, enzyme activities associated with the hydrolysis and oxidation of polysaccharides have increased. At the ecosystem scale, the biochemical characteristics of the dominant litter appear to modulate the effects of N deposition on organic matter dynamics.