The effects of municipal biosolids on nitrogen-fixing bacteria were assessed in a three month soil incubation study. Treatments included reference agricultural soils, soil amended with municipal biosolids or manure, and biosolids without soil. Nitrogen-fixation rates in reference and manure-amended soil were similar, and lower than in biosolids treatments; respiration rates showed similar trends. At test termination there was no difference between soil treatments for nitrogen-fixation, but some enhanced respiration in the biosolids-amended soils. Community structure was assessed using Biolog EcoPlates™ and denaturing gradient gel electrphoresis with a nitrogen-fixing gene (nifH). EcoPlate™ carbon utilization patterns corresponded with activity measures, with no difference among soil treatments at test termination. Nitrogen-fixing gene patterns showed a potential shift in community structure of biosolids-amended soils three months post-amendment. In general, the effects on the activity and structure of nitrogen-fixing communities were largely temporary; however, this study evaluated a one-time biosolids application. The potential for cumulative effects requires further investigation.