The idea of using constructed wetlands for the treatment and improving of wastewater emerged in the second half of the last century. Despite relatively wide use of this environmentally friendly technology, relatively little is known about the microbial populations involved in biotransformation and removal of contaminants in this system. The aim of the current study was to investigate the assembly and function of microbial populations in vertical-flow constructed wetland microcosms designed to improve the quality of wastewater after activated sludge treatment. Also, the performance of 3-year-old wetland ponds was investigated. Even though the quality of the influent water was relatively high, improvement in water parameters such as coliform level, ammonia concentration, BOD, and TSS was observed. The performance of the wetland ponds was comparable to that of the microcosms. The microbial community composition of the biofilm formed on the surface of gravel particles in vegetated and plant-free microcosms was studied by denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rRNA gene fragments. Highly complex bacterial diversity was observed in the biofilm. Cluster analysis of DGGE patterns demonstrated that depth within the wetland microcosm has a stronger effect on microbial community composition of the biofilm formed on wetland matrix than vegetation. Measurements of fluorescein diacetate hydrolysis activity and nitrification potential revealed that hydrolytic activity was affected by both microcosm depth and vegetation presence, whereas nitrification potential was mostly influenced by depth. Resolving the bacterial assemblage of wetland biofilm, which often is considered a black box, will help to understand the interactions involved in the development of diverse and mature biofilm and its function.