Almost ½ of the world’s land area comprises arid biomes where rainfall is very low (<500 mm/y) and potential evaporation is high. Sparsely-distributed freshwater systems in arid central Australia provide important refuges for mobile species and act as biotic reservoirs under adverse conditions. Some of these perennial systems are also evolutionary refugia that support relictual insects. Microbes play an important role in driving aquatic biogeochemical processes, but little is known about their composition and spatial patterns in and across these arid landscapes. Here we sought to assess the diversity and spatial distribution of aquatic Bacteria and Archaea in arid Australian waterbodies. We used 16s-rRNA gene amplicon sequencing to analyze the structure of microbial assemblages in biofilms and the water column from 8 waterbodies along a geographic axis across 2 drainage divisions. We used isotope hydrology to differentiate between groundwater-dominated waterbodies and those fed by surface water. We tested predictions and used metacommunity theory to explore spatial patterns between water column and biofilm microbiota, between perennial and temporary waterbodies, and between groundwater-fed and surface water-fed systems. We found greater similarity between assemblages of water column microbiota than those of the benthic biofilms. The spatial distributions of water column and biofilm assemblages were best explained by different metacommunity concepts. Distance decay matched the composition of the water column microbiota, whereas species sorting appeared to drive the composition of biofilm assemblages. The latter showed considerable within-site variability, changing with the type of benthic substrate present. Electrical conductivity and pH partly explained the composition of the water column and biofilm microbial assemblages, but only the water column microbiota was associated with the aquatic isotopic signatures. No distinctive microbiota were associated with perennial, groundwater-dominated sites previously identified as evolutionary refugia for aquatic insects. Temporary waterbodies contained significantly more heat and arid resilient microbial taxa commonly found in soils. Further investigation of temporal dynamics is now needed to determine how arid land aquatic microbiota might change under a warming climate.