We examined the spatiotemporal changes of microbial communities in relation to hydrochemistry variation over time and space in an aquifer polluted by landfill leachate (Banisveld, The Netherlands). Sampling in 1998, 1999, and 2004 at the same time of the year revealed that the center of the pollution plume was hydrochemically rather stable, but its upper fringe moved to the surface over time, especially at distances greater than 40 m away from the landfill. Complex and spatiotemporal heterogeneous bacterial and eukaryotic communities were resolved using denaturing gradient gel electrophoresis (DGGE) of 16S and 18S rRNA gene fragments. Large fluctuations were noted in the eukaryotic communities associated with strongly polluted and cleaner groundwater. The bacterial communities in strongly polluted samples were different from those in cleaner groundwater in 1998 and 1999, but no longer in 2004. The temporal variation in microbial communities was greater than the spatial variation: the 1998 bacteria communities in strongly polluted groundwater were more related to each other than to those recovered in 1999 and 2004. During the three sampling periods, the bacterial communities were more stable close to the landfill than at larger distances from the landfill. Overall, pollution appears to have only a minor influence on microbial communities. The considerable spatiotemporal variation in microbial community composition may contribute to better biodegradation of pollutants. Designing management strategies for natural attenuation of aquifer pollution will benefit from further long-term, high-density monitoring of changes in microbial communities, their diversity and physiological properties, in relation to changes in hydrochemistry.