A 2-year-old pilot-scale zerovalent iron (Fe-0)-permeable reactive barrier, treating groundwater contaminated with tetrachloroethylene and trichloroethylene, was microbially characterized using DNA-based polymerase chain reaction-DGGE (denaturing gradient gel electrophoresis) analysis. In situ mesocosm systems positioned in monitoring wells at different locations in the pilot-scale system allowed core sampling as a function of time without disturbing the barrier performance. Mesocosms were harvested from each location after 3, 7, and 23 months of operation and revealed the presence of Eubacteria, Archaea, and different functional groups of bacteria, which might affect iron barrier performance, including sulfate-reducing bacteria, iron(III)-reducing bacteria, denitrifying bacteria, and methanogens. DGGE fingerprints of the eubacterial 16S rDNA polymerase chain reaction amplification products clearly indicated different community structures in the iron material compared with the aquifer material. Gene sequencing of two dominant bands observed in samples collected from the iron barrier showed a maximum sequence similarity of 97% and 94% with a Firmicutes bacterium and an iron(III)-reducing bacterium enrichment culture clone, respectively. Results indicate that Fe-0 had a profound impact on the microbial community composition, most likely by decreasing the redox potential and increasing the pH of the groundwater.