Biological treatment using sulfate-reducing bacteria (SRB) is a promising approach to remediate acid rock drainage (ARD). Our purpose was to assess the performance of a sequential system consisting of a limestone bed filter followed by a sulfate-reducing bioreactor treating synthetic ARD for 375 days and to evaluate changes in microbial composition. The treatment system was effective in increasing the pH of the ARD from 2.7 to 7.5 and removed total Cu(II) and Zn(II) concentrations by up to 99.8% and 99.9%, respectively. The presence of sulfate in ARD promoted sulfidogenesis and changed the diversity and structure of the microbial communities. Methansarcina spp. was the most abundant amplicon sequence variant (ASV); however, methane production was not detected. Biodiversity indexes decreased over time with the bioreactor operation, whereas SRB abundance remained stable. Desulfobacteraceae, Desulfocurvus, Desulfobulbaceae and Desulfovibrio became more abundant, while Desulfuromonadales, Desulfotomaculum and Desulfobacca decreased. Geobacter and Syntrophobacter were enriched with bioreactor operation time. At the beginning, ASVs with relative abundance <2% represented 65% of the microbial community and 21% at the end of the study period. Thus, the results show that the microbial community gradually lost diversity while the treatment system was highly efficient in remediating ARD.