To better understand dissimilatory iron and sulfate reduction (DIR and DSR) by subsurface microorganisms, we investigated the effects of sulfate and electron donors on the microbial goethite (α-FeOOH) reduction. Batch systems were created 1) with acetate or glucose (donor), 2) with goethite and sulfate (acceptor), and 3) with aquifer sediment (microbial source). With 0.2 mM sulfate, goethite reduction coupled with acetate oxidation was limited. However, with 10 mM sulfate, 8 mM goethite reduction occurred with complete sulfate reduction and x-ray absorption fine-structure analysis indicated the formation of iron sulfide. This suggests that goethite reduction was due to the sulfide species produced by DSR bacteria rather than direct microbial reaction by DIR bacteria. Both acetate and glucose promoted goethite reduction. The rate of goethite reduction was faster with glucose, while the extent of goethite reduction was higher with acetate. Sulfate reduction (10 mM) occurred only with acetate. The results suggest that glucose-fermenting bacteria rapidly stimulated goethite reduction, but acetate-oxidizing DSR bacteria reduced goethite indirectly by producing sulfides. This study suggests that the availability of specific electron donor and sulfate significantly influence microbial community activities as well as goethite transformation, which should be considered for the bioremediation of contaminated environments.