Groundwater microbial community samples are traditionally collected using pumping techniques optimized for groundwater chemistry assessment, although the impact of groundwater pumping parameters on apparent bacterial community structures (BCSs) is not really known. We therefore studied the impact of pumping lift, flow regime, and tubing material on BCS, which were analyzed by terminal-restriction fragment length polymorphism (T-RFLP). Ruzicka dissimilarity coefficients were calculated between T-RFLP profiles to assess disparities between BCS. Variations in pumping lift, flow regime, and tubing material did not affect the apparent BCS in experiments using a homogenous water system under laboratory conditions showing that the conditions within the tube had no detectable effect on BCS. However, pumping groundwater from aquifer monitoring wells at different flow rates in the field revealed a significant impact on the apparent BCS. Water samples collected from fine sediment were the most affected by the pumping flow rate.