Denitrification is an anaerobic microbial process that transforms nitrate (NO3−) to nitrogen (N) gas, preventing the movement of NO3− into coastal waters where it can lead to eutrophication. Urbanization can reduce the potential for denitrification in riparian zones and streams by altering the environmental conditions that foster denitrification (i.e., low oxygen and available C). Here we evaluated the factors limiting denitrification potential in forested and herbaceous riparian and stream pool and organic debris dam habitats in urban, suburban, exurban, and forested reference watersheds in the Baltimore, Maryland metropolitan area. Denitrification potential (with and without C and NO3− additions) and microbial biomass C and N content, potential net N mineralization and nitrification, microbial respiration, and inorganic N pools were measured in summer (June) and fall (November). Denitrification potentials were highest in the herbaceous riparian soils and lowest in pool sediments. Forested riparian soil denitrification potential was highest in the exurban watershed but in other habitats did not vary with watershed type. Nearly all variables were higher in June than in November. Overall, C was a more important driver of denitrification potential than N; potentials in unamended and N-amended treatments were very similar (<200 ng N>g−1 h−1) and were much lower than in the C-amended and C+N-amended treatments (>800 ng N g−1 h−1). Our results suggest that efforts to enhance denitrification in urban watersheds need to focus on the differential controls of denitrification across habitats, urban land use types, and seasons.