Many studies have shown that changes in nitrogen (N) availability affect primary productivity in a variety of terrestrial systems, but less is known about the effects of the changing N cycle on soil organic matter (SOM) decomposition. We used a variety of techniques to examine the effects of chronic N amendments on SOM chemistry and microbial community structure and function in an alpine tundra soil. We collected surface soil (0–5 cm) samples from five control and five long-term N-amended plots established and maintained at the Niwot Ridge Long-term Ecological Research (LTER) site. Samples were bulked by treatment and all analyses were conducted on composite samples. The fungal community shifted in response to N amendments, with a decrease in the relative abundance of basidiomycetes. Bacterial community composition also shifted in the fertilized soil, with increases in the relative abundance of sequences related to the Bacteroidetes and Gemmatimonadetes, and decreases in the relative abundance of the Verrucomicrobia. We did not uncover any bacterial sequences that were closely related to known nitrifiers in either soil, but sequences related to archaeal nitrifiers were found in control soils. The ratio of fungi to bacteria did not change in the N-amended soils, but the ratio of archaea to bacteria dropped from 20% to less than 1% in the N-amended plots. Comparisons of aliphatic and aromatic carbon compounds, two broad categories of soil carbon compounds, revealed no between treatment differences. However, G-lignins were found in higher relative abundance in the fertilized soils, while proteins were detected in lower relative abundance. Finally, the activities of two soil enzymes involved in N cycling changed in response to chronic N amendments. These results suggest that chronic N fertilization induces significant shifts in soil carbon dynamics that correspond to shifts in microbial community structure and function.