This EnviroAtlas dataset contains data on the mean biological nitrogen fixation in natural/semi-natural ecosystems per 12-digit Hydrologic Unit (HUC) in 2006. Biological N fixation (BNF) in natural/semi-natural ecosystems was estimated using a correlation with actual evapotranspiration (AET). This correlation is based on a global meta-analysis of BNF in natural/semi-natural ecosystems (Cleveland et al. 1999). AET estimates for 2006 were calculated using a regression equation describing the correlation of AET with climate (average annual daily temperature, average annual minimum daily temperature, average annual maximum daily temperature, and annual precipitation) and land use/land cover variables in the conterminous US (Sanford and Selnick 2013). Data describing annual average minimum and maximum daily temperatures and total precipitation for 2006 were acquired from the PRISM climate dataset (http://prism.oregonstate.edu). Average annual climate data were then calculated for individual 12-digit USGS Hydrologic Unit Codes (HUC12s; https://water.usgs.gov/GIS/huc.html; 22 March 2011 release) using the Zonal Statistics tool in ArcMap 10.0. AET for individual HUC12s was estimated using equations described in Sanford and Selnick (2013). BNF in natural/semi-natural ecosystems within individual HUC12s was modeled with an equation describing the statistical relationship between BNF (kg N ha-1 yr-1) and actual evapotranspiration (AET; cm yr-1) and scaled to the proportion of non-developed and non-agricultural land in the HUC12. The first half of the equation represents the most conservative estimate in a meta-analysis of BNF in natural/semi-natural ecosystems (Cleveland et al. 1999), and was chosen over the central and high estimates because recent, top-down global mass balances that suggest that natural BNF rates are less than previous estimates based on scaled-up estimates from individual plots (Vitousek et al. 2013). The land use/land cover modifier is not included in the original Cleveland et al. (1999) analysis. We believe it is appropriate to include so as not to overestimate BNF in HUC12s with large proportions of urban or agricultural development. These data represent an average N input to individual HUC12s, i.e., they are not specific to an individual land use type within the HUC12. This dataset was produced by the US EPA to support research and online mapping activities related to EnviroAtlas. EnviroAtlas (https://www.epa.gov/enviroatlas) allows the user to interact with a web-based, easy-to-use, mapping application to view and analyze multiple ecosystem services for the contiguous United States. The dataset is available as downloadable data (https://edg.epa.gov/data/Public/ORD/EnviroAtlas) or as an EnviroAtlas map service. Additional descriptive information about each attribute in this dataset can be found in its associated EnviroAtlas Fact Sheet (https://www.epa.gov/enviroatlas/enviroatlas-fact-sheets).