Anthropogenic activities have significantly altered global biogeochemical nitrogen (N) cycling leading to major environmental problems including freshwater eutrophication. Soils in the riparian zone, the interface between terrestrial and aquatic ecosystems, may decrease N loads to streams through plant uptake and microbial transformations. However, the ecological functioning of riparian zones are often compromised due to degraded physical and biological conditions (e.g. vegetation clearing, invasive species, and nutrient pollution). Restoration to maximise N retention in riparian zones requires a mechanistic understanding of the processes which underpin the microbial N cycle, particularly the spatial distribution of N and the structure and activity of key N-cycling microbes. Our aim was to assess the spatial organisation of soil microbial communities in riparian zones of contrasting land use (agricultural versus native vegetation ecosystems) using 16S sequencing accompanied with qPCR of archaeal and bacterial nitrogen cycling functional genes (AOA, AOB, nirK, nirS, nosZ). Riparian soil was sampled at 0m (parafluvial zone), 1m, 2m, 5m and 10m distance from one stream within each land use with four latitudinal transects each. Riparian vegetation cover was characterised to measure localised disturbance. Soil physiochemistry (TOC, NH4+, N03-), N functional genes and microbial community composition differed between land uses and by distance from the stream. All N functional genes were more abundant at the native site, particularly in the parafluvial zone, likely owing to greater diversity of vegetation habitats and soil physiochemistry compared to the agricultural site. The abundance of nirS and nosZ, key enzymes in the soil N denitrification pathyway, were highest in the native ecosystem parafluvial zone. AOA, nirS and nosZ all increased towards the stream at both sites; indicative of an increase in microbial activity in areas with greater organic deposition and fluctuating anoxic/oxic conditions (due to intermittent inundation). There was no latitudinal pattern for AOB or nirK. These findings highlight the importance of parafluvial soils as catalysts for N processing, especially in stream reaches dominated by native vegetation. Spatial partitioning of N fractions and microbial communities in riparian zones should be accounted for when planning restoration activities.