Hypersaline ecosystems—aquatic environments where concentration of salt exceeds 35 g L−1—host microbial communities that are highly specialised to cope with these extreme conditions. However, our knowledge on the taxonomic diversity and functional metabolisms characterising microbial communities in the water columns of hypersaline ecosystems is still limited, and this may compromise the future preservation of these unique environments. DNA metabarcoding provides a reliable and affordable tool to investigate environmental dynamics of aquatic ecosystems, and its use in brine can be highly informative. Here, we make use of bacterial 16S metabarcoding techniques combined with hydrochemical analyses to investigate the microbial patterns (diversity and functions) from five hypersaline lakes located at Rottnest Island (WA). Our results indicate lake-driven microbial aquatic assemblages that are characterised by taxonomically and functionally moderately to extremely halophilic groups, with TDS (total dissolved solids) and alkalinity amongst the most influential parameters driving the community patterns. Overall, our findings suggest that DNA metabarcoding allows rapid but reliable ecological assessment of the hypersaline aquatic microbial communities at Rottnest Island. Further studies involving different hypersaline lakes across multiple seasons will help elucidate the full extent of the potential of this tool in brine.