Global water supplies are threatened by climate changes and the expansion of urban areas, which have led to an increasing interest in nature-based solutions for water reuse and reclamation. Reclaimed water is a possible resource for recharging aquifers, and the addition of an organic reactive barrier has been proposed to improve the removal of pollutants. There has been a large focus on organic pollutants, but less is known about multifunctional barriers, that is, how barriers also remove nutrients that threaten groundwater ecosystems. Herein, we investigated how compost- and woodchip-based barriers affect nitrogen (N) removal in a pilot soil aquifer treatment facility designed for removing nutrients and recalcitrant compounds by investigating the composition of microbial communities and their capacity for N transformations. Secondary-treated, ammonium-rich wastewater was infiltrated through the barriers, and the changes in the concentration of ammonium, nitrate, and dissolved organic carbon (DOC) were measured after passage through the barrier during 1 year of operation. The development and composition of the microbial community in the barriers were examined, and potential N-transforming processes in the barriers were quantified by determining the abundance of key functional genes using quantitative PCR. Only one barrier, based on compost, significantly decreased the ammonium concentration in the infiltrated water. However, the reduction of reactive N in the barriers was moderate (between 21 and 37%), and there were no differences between the barrier types. All the barriers were after 1 year dominated by members of Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria, although the community composition differed between the barriers. Bacterial classes belonging to the phylum Chloroflexi showed an increased relative abundance in the compost-based barriers. In contrast to the increased genetic potential for nitrification in the compost-based barriers, the woodchip-based barrier demonstrated higher genetic potentials for denitrification, nitrous oxide reduction, and dissimilatory reduction of nitrate to ammonium. The barriers have previously been shown to display a high capacity to degrade recalcitrant pollutants, but in this study, we show that most barriers performed poorly in terms of N removal and those based on compost also leaked DOC, highlighting the difficulties in designing barriers that satisfactorily meet several purposes.