While millions of people drink arsenic-contaminated tube well water across Bangladesh, there is no recent scientific explanation which is able to either comprehensively explain arsenic mobilization or to predict the spatial distribution of affected wells. Rather, mitigation strategies have focused on the sinking of deep tube wells into the currently arsenic-free Pleistocene aquifer. In this study, Bangladesh shallow tube wells identified as contaminated and uncontaminated, as well as deep tube wells, were analyzed for geochemical and in situ microbiological composition. Whereas arsenic was detected in all Holocene aquifer wells, no arsenic was found in wells accessing the Pleistocene aquifer. Bacterial genera, including Comamonadaceae, Acidovorax, Acinetobacter, and Hydrogenophaga, associated with tolerance of high arsenic concentrations, rather than dissimilatory Fe(III) or As(V) reduction, were identified in shallow tube wells, indicating that mobilization may not occur at depth, but is rather due to drawdown of surface contaminated water. Deep tube wells contained microbes indicative of aerobic conditions, including the genera Aquabacterium, Limnobacter, and Roseomonas. It is concluded that through drawdown of arsenic or organic matter, further utilization of the Pleistocene aquifer could result in contamination similar to that observed in the Holocene aquifer.