Acid mine drainage (AMD) is a serious environmental problem worldwide that requires efficient and sustainable remediation technologies including the use of biological mechanisms. A key challenge for AMD bioremediation is to provide optimal conditions for microbial-mediated immobilisation of trace metals. Although organic carbon and oxygen can enhance treatment efficiency, the effect on microbial communities is unclear. In this study, surface sediments from a natural wetland with proven efficiency for AMD bioremediation were artificially exposed to oxygen (by aeration) and/or organic carbon (in the form of mixed organic acids) and incubated under laboratory conditions. In addition to measuring changes in water chemistry, a metagenomics approach was used to determine changes in sediment bacterial, archaeal and fungal community structure, and functional gene abundance. The addition of organic carbon produced major changes in the abundance of microorganisms related to iron and sulfur metabolism (including Geobacter and Pelobacter) and increased levels of particulate metals via sulfate reduction. Aeration resulted in an increase in Sideroxydans abundance but no significant changes in metal chemistry were observed. The study concludes that the utilisation of organic carbon by microorganisms is more important for achieving efficient AMD treatment than the availability of oxygen, yet the combination of oxygen with organic carbon addition did not inhibit the improvements to water quality