Passive bioremediation of metal- and sulfate-containing acid mine drainage (AMD) has been investigated in a batch study. Multiple substrates were used in the AMD remediation using spent mushroom compost (SMC), limestone, activated sludge (AS), and woodchips (WC) under anoxic conditions suitable for bacterial sulfate reduction (BSR). Limestones used were of crushed limestone (CLS) and uncrushed limestone, provided at two different ratios in mixed substrates treatment and varied by the proportion of SMC and limestone. The SMC greatly assisted the removals of sulfate and metals and also acted as an essential carbon source for BSR. The mixed substrate composed of 40% CLS, 30% SMC, 20% AS, and 10% WC was found to be effective for metal removal. Mn, Cu, Pb, and Zn were greatly removed (89-100%) in the mixed substrates treatment, while Fe was only removed at 65%. Mn was found to be removed at a greatly higher rate than Fe, suggesting important Mn adsorption onto organic materials, that is, greater sorption affinity to the SMC. Complementary with multiple treatment media was the main mechanism assisting the AMD treatment through microbial metal reduction reactions.