<p>Neutral and metalliferous drainage (NMD) poses an environmental risk for both operating and legacy mine sites. Near-neutral pH distinguishes NMD from more acidic conditions of acid and metalliferous drainage (AMD), however NMD contains elevated levels of metals that necessitate strict management. Microbial communities are key indicators of ecological conditions and play important roles in NMD biogeochemical cycling, often exhibiting distinct dynamics compared to AMD. Shotgun sequencing and metagenome assembled genomes (MAGs) were used to characterize microbial diversity and functional potential across water, biofilm, and sediment microbiomes along a flow path at a historical lead–zinc mine in Western Australia. Zn levels peaked upstream and declined downstream, corresponding to shifts in microbial diversity. In water microbiomes, a <i>Polynucleobacter</i> MAG became dominant where Zn concentrations dropped below known toxicity thresholds. The genomic traits of <i>Polynucleobacter</i>, including a streamlined genome, Zn- (<i>LpxC</i>) and heat-responsive membrane genes, and enriched lipid metabolism pathways, enabled survival under metal and nutrient stress. Photosynthetic biofilms, dominated by cyanobacteria such as <i>Synechococcaceae</i> and <i>Leptolyngbyaceae</i>, played a central role in ecosystem function. These biofilms contained genes for photosynthesis, metal transport, and motility, and likely contributed organic carbon and sulfur intermediates that supported heterotrophs like <i>Polynucleobacter</i> and <i>Sediminibacterium</i>. Coordinated microbial sulfur metabolism across habitats was evident, with sulfur oxidation occurring in water and biofilms and sulfate reduction localized to sediment, evidenced with ZnS mineral phases associated with increased <i>Dsr</i>MKJOP gene abundance. These findings are vital for mine closure and land reclamation, offering knowledge on key microbial adaption and syntrophy in NMD systems.</p> Graphical abstract <p></p>