Field experiments were conducted over a 460-day period to assess the efficiency of different mixtures of organic substrates to remediate coalmine-generated acid mine drainage (AMD). Five pilot-scale, flow-through bioreactors containing mixtures of herbaceous and woody organic substrates along with one control reactor containing only limestone were constructed at the Tab-Simco site and exposed to AMD in situ. Tab-Simco is an abandoned coal mine near Carbondale, Illinois that produces AMD with pH ∼2.5 and notably high average concentrations of SO4 (5050 mg/L), Fe (950 mg/L), Al (200 mg/L), and Mn (44 mg/L). Results showed that the sequestration of SO4 and metals was achieved in all reactors; however, the presence and type of organic carbon matrix impacted the overall system dynamics and the AMD remediation efficiency. All organic substrate-based reactors established communities of sulfate reducing microorganisms that contributed to enhanced removal of SO4, Fe, and trace metals (i.e., Cu, Cd, Zn, Ni) via microbially-mediated reduction followed by precipitation of insoluble sulfides. Additional mechanisms of contaminant removal were active in all reactors and included Al- and Fe-rich phase precipitation and contaminant surface sorption on available organic and inorganic substrates. The organic substrate-based reactors removed more SO4, Fe, and Al than the limestone-only control reactor, which achieved an average removal of ∼19 mol% SO4, ∼49 mol% Fe, 36 mol% Al, and 2 mol% Mn. In the organic substrate-based reactors, increasing herbaceous content correlated with increased removal efficiency of SO4 (26–35 mol%), Fe (36–62 mol%), Al (78–83 mol%), Mn (2–6 mol%), Ni (64–81 mol%), Zn (88–95 mol%), Cu (72–85 mol%), and Cd (90–92 mol%), while the diversity of the intrinsic microbial community remained relatively unchanged. The extrapolation of these results to the full-scale Tab-Simco treatment system indicated that, over the course of a 460-day period, the predominantly herbaceous bioreactors could remove up to 92,500 kg SO4, 30,000 kg Fe, 8,950 kg Al, and 167 kg Mn, which represents a 18.3 wt%, 36.8 wt%, 4.1 wt% and 82.3 wt% increase in SO4, Fe, Al, and Mn, respectively, removal efficiency compared to the predominantly ligneous bioreactors.The results imply that anaerobic organic substrate bioreactors are promising technologies for remediation of coal-generated AMD and that increasing herbaceous content in the organic substrate matrix can enhance contaminant sequestration. However, in order to improve the remediation capacity, future designs must optimize not only the organic carbon substrate but also include a pretreatment phase in which the bulk of dissolved Fe/Al-species are removed from the influent AMD prior to entering the bioreactor because of 1) seasonal variations in temperature and redox gradients could induce dissolution of the previously formed redox sensitive compounds, and 2) microbially-mediated sulfate reduction activity may be inhibited by the excessive precipitation of Al- and Fe-rich phases.