Devils Hole is the sole natural habitat of the critically endangered Devils Hole pupfish (Cyprinodon diabolis). To establish a backup population, the Ash Meadows Fish Conservation Facility (AMFCF), a full-scale replica of the uppermost 6.7 m of Devils Hole, was constructed by management agencies in the mid-2010s. Despite rigorous efforts to mimic the bathymetric and physical details of the Devils Hole environment, the biogeochemistry and microbiology of the AMFCF refuge tank remain largely unaddressed. We evaluated water physicochemistry and employed Illumina DNA sequencing of 16S rRNA gene libraries to evaluate planktonic and benthic bacterial and archaeal community composition within their respective physicochemical contexts in Devils Hole and AMFCF on the same day. Major ion concentrations were consistent between the two systems, but water temperature and dissolved oxygen dynamics differed. Bioavailable nitrogen (primarily nitrate) was 5x lower in AMFCF. Devils Hole and AMFCF nitrogen:phosphorus molar ratios were 107:1 and 22:1, indicative of different nutrient control mechanisms. Both sites are microbiologically diverse, with over 40 prokaryotic phyla represented at each, with 37 shared between them and nearly than half deriving from candidate divisions. The abundance and composition of predicted photosynthetic primary producers (Cyanobacteria) was markedly different between sites: Devils Hole planktonic and sediment communities were dominated by Oscillatoria spp. (13.2% mean relative abundance), which proved virtually undetectable in AMFCF. Conversely, AMFCF was dominated by a predicted heterotroph from the Verrucomicrobiaceae family (31.7%); which was comparatively rare (<2.4%) in Devils Hole. We propose that the paucity of bioavailable nitrogen in AMFCF, perhaps resulting from physical isolation from allochthonous environmental inputs, is reflected in the microbial assemblage disparity, influences biogeochemical cycling of other dissolved constituents, and may ultimately impact survivorship and recruitment of refuge populations of the Devils Hole pupfish.