Urban expansion causes coastal wetland loss, and environmental stressors associated with development can lead to wetland degradation and loss of ecosystem services. This study investigated the effect of urbanization on prokaryotic community composition in tidal freshwater wetlands. Sites in an urban, suburban, and rural setting were located near Buenos Aires, Argentina, and Washington D.C., USA.We sampled soil associated with two pairs of functionally similar plant species, and used Illumina sequencing of the 16S rRNA gene to examine changes in prokaryotic communities. Urban stressors included raw sewage inputs, nutrient pollution, and polycyclic aromatic hydrocarbons. Prokaryotic communities changed along the gradient (nested PerMANOVA, Buenos Aires: p = 0.005; Washington D.C.: p = 0.001), but did not differ between plant species within sites. Indicator taxa included Methanobacteria in rural sites, and nitrifying bacteria in urban sites, and we observed a decrease in methanogens and an increase in ammonia-oxidizers from rural to urban sites. Functional profiles in the Buenos Aires communities showed higher abundance of pathways related to nitrification and xenobiotic degradation in the urban site. These results suggest that changes in prokaryotic taxa across the gradient were due to surrounding stressors, and communities in urban and rural wetlands are likely carrying out different functions.