The microbial communities of high-latitude ecosystems are expected to experience rapid changes over the next century due to climate warming and increased deposition of reactive nitrogen, changes that will likely affect microbial community structure and function. In moist acidic tundra (MAT) soils on the North Slope of the Brooks Range, Alaska, substantial losses of C and N were previously observed after long-term nutrient additions. To analyse the role of microbial communities in these losses, we utilized 16S rRNA gene tag pyrosequencing coupled with community-level physiological profiling to describe changes in MAT bacterial communities after short- and long-term nutrient fertilization in four sets of paired control and fertilized MAT soil samples. Bacterial diversity was lower in long-term fertilized plots. The Acidobacteria were one of the most abundant phyla in all soils and distinct differences were noted in the distributions of Acidobacteria subgroups between mineral and organic soil layers that were also affected by fertilization. In addition, Alpha- and Gammaproteobacteria were more abundant in long-term fertilized samples compared with control soils. The dramatic increase in sequences within the Gammaproteobacteria identified as Dyella spp. (order Xanthomonadales) in the long-term fertilized samples was confirmed by quantitative PCR (qPCR) in several samples. Long-term fertilization was also correlated with shifts in the utilization of specific substrates by microbes present in the soils. The combined data indicate that long-term fertilization resulted in a significant change in microbial community structure and function linked to changes in carbon and nitrogen availability and shifts in above-ground plant communities.