The microbial population of a sludge amended leaf compost material utilized for treatment of artificial acid mine drainage was studied by culture-independent molecular methods. Iron-rich and sulfurous wastewater (artificial acid mine drainage) was circulated through a column bioreactor for 16 months. After 12 months the column was inoculated with a mixed culture from an acidic pond receiving acid mine drainage from a tailings impoundment at a decommissioned site in Kristineberg, North Sweden. Hydrogen sulfide odor and the formation of black precipitates indicated that sulfate-reduction occurred in the column. 16S rDNA gene analysis by denaturing gradient gel electrophoresis, cloning, and sequencing as well as fluorescent in situ hybridization confirmed the presence of microorganisms closely related to sulfate-reducing bacteria and microorganisms from the genera Pseudoxanthmonas, Dechlorosoma, Desulfovibrio, Agrobacterium, Methylocapsa, Rhodococcus, Sulfobacillus, and some unidentified bacteria. Sulfate-reducing bacteria were found in the column bioreactor 2 weeks after inoculation, but not thereafter. This suggests they were in low abundance, even though sulfate remediation rates were significant. Instead, the population contained species similar to those previously found to utilize humic substances released from the compost material.