This study compared the efficiency of five emergent plant species (Carex elata All., Juncus effusus L., Typhoides arundinacea (L.) Moench var. picta, Phragmites australis (Cav.) Trin. and Typha latifolia L.) used for the decontamination of nitrogen polluted water at mesoscale level. The correlations between N removal and microorganisms content and activity on root and gravel surfaces were also evaluated in order to broaden the knowledge about water purification processes. The experiment was conducted in plastic tanks for two years (2008–2009), performing drying/wetting application cycles with NH4NO3 solution. At the end of the experiment, root and gravel samples were collected from each tank for the measurement in aqueous extracts of total cultivable microbial population and Pseudomonas genus, and -glucosidase, chitinase, phosphatase and leucine aminopeptidase activities; other base chemical analyses (electrical conductivity, water soluble carbon, nitrate and ammonia) were conducted. The production of bio-available root exudates was evaluated though the quantification of water soluble carbon (WSC) in root and gravel washing water. WSC seems to support the plant denitrification activity because T. arundinacea and Ph. australis systems, which showed the highest WSC content, achieved the highest denitrification percentages (37% and 34%, respectively). In these plant systems the higher cultivable microbial population and rhizospheric hydrolytic activity found were probably induced by the high content of soluble organic substrates that derived from root turnover and root exudation, without addition of any other carbon source. The establishment of this active micro-environment (rhizo-biospace) has high ecological significance due to the role played by microbial activity in the nutrient cycles and is of great importance when choosing suitable plant species for wastewater treatment.