The groundwater biome is a poorly characterized habitat hypothesized to harbor uniquely diverse bacterial communities; the degree to which these communities differ from associated soils is a central question in environmental microbiology. We characterized the Bacterial community composition in 37 aquifer and 32 surface soil samples across the island of O'ahu, Hawai'i. Several bacterial phyla (Acetothermia, Omnitrophica, Parcubacteria, Peregrinibacteria) relatively abundant in the aquifer samples were rare to absent in the soils. Immense bacterial diversity detected in the deep aquifers indicates that these environments are not as homogenous as expected, but provide various niches and energy sources for wide variety of bacteria. A small proportion of OTUs were widespread in all the basal (0.63%) and all the dike aquifer (0.31%) samples. However, these core bacteria comprised an average of 31.8% (ranging 16.2%-62.0%) and 15.4% (0.1%-31.5%) of all sequences isolated from the basal and dike aquifers respectively. Bacterial community composition correlated significantly with the sodium, sulfate, potassium, total dissolved solids, nitrate, conductivity, and pH in the basal aquifers, while phosphate and bicarbonate levels were also highly important when dike water samples were included in the analyses. This was consistent with high relative abundance of putative chemolithoautoroph taxa in the aquifer communities relative to soils. Targeted molecular and culture-based fecal indicator microbial analyses indicated good water quality of aquifers. The dominance of unique, deeply branching lineages in tropical aquifers emphasizes a large adaptive potential in O'ahu's aquifers; variability among groundwater samples suggests that aquifer habitats are surprisingly variable potentially harboring a variety of chemolithotrophic energy sources. Although parallel analyses of conventional and alternative indicators indicated good groundwater quality, this study calls for groundwater monitoring programs which would consider public as well as ecosystem health.