Anthropogenic nitrogen (N) emissions have caused an increase in global N deposition, and the northeastern United States is a N deposition hotspot. Nitrogen deposition could impact soil microbial abundance because most soil biota evolved under low N conditions. Soil microbes are key to many ecosystem processes such as organic matter decomposition, which makes nutrients available for plant growth. Therefore, understanding the factors controlling microbial abundance is critical for being able to predict ecosystem processes. We hypothesized that fungal and bacterial abundance and the fungi:bacteria ratio in soil would decrease along a natural N deposition gradient in the northeastern US, and be further reduced by fertilizer application. To address our hypothesis, we collected soil from eight different sites, six of which had experimental plots where N fertilizer had been added. At each site, we collected soil from four ambient or control plots and, if available, four fertilized plots (48 total plots). We measured microbial abundance using phospholipid fatty acid analysis (PLFA). We compared microbial abundance in fertilized to control plots using t-tests, and we analyzed changes in microbial abundance along the natural N gradient using regression analysis. We found that ambient N reduced bacterial abundance (p = 0.03, R2 = 0.08), but it did not affect fungal abundance (p = 0.17, R2 = 0.02). Fertilizer had no overall effect on fungal (p = 0.72, t = 0.36) or bacterial abundance (p = 0.91, t = -0.12). However, fertilizer did reduce bacterial (p = 0.02, F = 5.6) and fungal (p = 0.02, F = 5.8) abundance at high levels of ambient N deposition. In conclusion, our study showed that soil microbial responses to fertilizer in the northeastern US depend on the background levels of atmospheric N deposition.