Significance Biological nitrogen fixation (BNF) is the largest natural source of new nitrogen (N) to terrestrial ecosystems. Tropical forest ecosystems are a putative global hotspot of BNF, but direct, spatially explicit measurements in the biome are virtually nonexistent. Nonetheless, robust estimates of tropical forest BNF are critical for understanding how these important ecosystems may respond to global change and assessing human perturbations to the N cycle. Here, we introduce a spatial sampling method to assess BNF and present evidence that tropical forest BNF is much lower than previously assumed. Our results imply that humans have roughly doubled N inputs to the tropical forest biome relative to N inputs through BNF.