Zero-valent iron (Fe0)-based permeable reactive barrier treatment has been generating great interest for passive groundwater remediation, yet few studies have paid particular attention to the microbial activity and characteristics within and in the vicinity of the Fe0-barrier matrix. The present study was undertaken to evaluate the microbial population and community composition in the reducing zone of influence by Fe0 corrosion in the barrier at the Oak Ridge Y-12 Plant site. Both phospholipid fatty acids and DNA analyses were used to determine the total microbial population and microbial functional groups, including sulfate-reducing bacteria, denitrifying bacteria, and methanogens, in groundwater and soil/iron core samples. A diverse microbial community was identified in the strongly reducing Fe0 environment despite a relatively high pH condition within the Fe0 barrier (up to pH approximately 10). In comparison with those found in the background soil/groundwater samples, the enhanced microbial population ranged from approximately 1 to 3 orders of magnitude and appeared to increase from upgradient of the barrier to downgradient soil. In addition, microbial community composition appeared to change over time, and the bacterial types of microorganisms increased consistently as the barrier aged. DNA analysis indicated the presence of sulfate-reducing and denitrifying bacteria in the barrier and its surrounding soil. However, the activity of methanogens was found to be relatively low, presumably as a result of the competition by sulfate/metal-reducing bacteria and denitrifying bacteria because of the unlimited availability of sulfate and nitrate in the site groundwater. Results of this study provide evidence of a diverse microbial population within and in the vicinity of the iron barrier, although the important roles of microbial activity, either beneficially or detrimentally, on the longevity and enduring efficiency of the Fe0 barriers are yet to be evaluated.