Relatively little is known regarding the spatial variability of microbial communities in aquifers where well fouling is an issue. In this study 2 water wells were installed in an alluvial aquifer located adjacent to the North Saskatchewan River and an associated piezometer network developed to facilitate the study of microbial community structure, richness, and diversity. Carbon utilization data analysis revealed reduced microbial activity in waters collected close to the wells. Functional PCR and quantitative PCR analysis indicated spatial variability in the potential for iron-, sulphate-, and nitrate-reducing activity at all locations in the aquifer. Denaturing gradient gel electrophoresis analysis of aquifer water samples using principal components analyses indicated that the microbial community composition was spatially variable, and denaturing gradient gel electrophoresis sequence analysis revealed that bacteria belonging to the genera Acidovorax , Rhodobacter , and Sulfuricurvum were common throughout the aquifer. Shannon's richness (H') and Pielou's evenness (J') indices revealed a varied microbial diversity (H' = 1.488-2.274) and an even distribution of microbial communities within the aquifer (J' = 0.811-0.917). Overall, these analyses revealed that the aquifer's microbial community varied spatially in terms of composition, richness, and metabolic activity. Such information may facilitate the diagnosis, prevention, and management of fouling.