Bundera sinkhole represents Australia’s only deep anchialine environment: an inland oligohaline pool connected to the ocean via a lengthy subterranean cave. Such habitats generally contain a rich diversity of micro- and macrooganisms, many unique to anchialine environments, yet few studies have examined the microbial component of such systems in depth. The water column within the Bundera sinkhole was sampled at multiple depths, from 2m down to 28m to collect biological material and measure chemical and physical parameters. We performed I6S rRNA gene amplicon and metagenomic sequencing to investigate the ecology, structure and metabolic capabilities of the residing microbial communities. Our results show that this environment supports a series of very distinct microbial assemblages at different depths. Community composition can be correlated with a number of different physicochemical parameters. Representatives of sulphur-oxidizing, nitrifying and ammonia-oxidizing organisms in samples below 8 m provide us with new insight into the variety of chemolithotrophic primary production mechanisms employed in this interesting ecosystem, the only known occurrence in the Southern Hemisphere.