A multiphasic study using structural and functional analyses was employed to investigate the spatial dynamics of the microbial community within five horizontal subsurface flow treatment wetlands (TWs) of differing designs in Germany. The TWs differed in terms of the depth of media saturation, presence of plants (Phragmites australis), and aeration. In addition to influent and effluent water samples, internal samples were taken at different locations (12.5 %, 25 %, 50 %, and 75 % of the fractional distance along the flow path) within each system. 16S rRNA sequencing was used for the investigation of microbial community structure and was compared to microbial community function and enumeration data. The microbial community structure in the unaerated systems was similar, but different from the aerated TW profiles. Spatial positioning along the flow path explained the majority of microbial community dynamics/differences within this study. This was mainly attributed to the availability of nutrients closer to the inlet which also regulated the fixed biofilm/biomass densities. As the amount of fixed biofilm decreased from the inlet to the TW outlets, structural diversity increased, suggesting different microbial communities were present to handle the more easily utilized/degraded pollutants near the inlet vs. the more difficult to degrade and recalcitrant pollutants closer to the outlets. This study also confirmed that effluent water samples do not accurately describe the microbial communities responsible for water treatment inside a TW, highlighting the importance of using internal samples for investigating microbial communities in TWs. The results of this study reinforce an existing knowledge gap regarding the potential for TW design modifications which incorporate microbial community spatial dynamics (heterogeneity). It is suggested that utilizing step-feeding could allow for improved water treatment within the same areal footprint, and modifications enhancing co-metabolic processes could assist in improving the treatment of more difficult to degrade or recalcitrant compounds such as micropollutants.