Although two species of chemolithoautotrophic bacteria, Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, are widely considered to be the main microorganisms that control the process of bioleaching of heavy metals from sewage sludge, little is known about the effect of dissolved organic matter (DOM) present in sewage sludge on bacterial oxidation of energy substrate. Batch cultures studies showed that sludge DOM significantly inhibited ferrous iron and sulfur oxidation by Acidithiobacillus ferrooxidans LX5 and Acidithiobacillus thiooxidans TS6, respectively. The toxicity of sludge DOM appeared when the concentration was higher than 150 mg DOC L−1. Among the organic compounds tested, the monocarboxylic organic acids including formic acid, acetic acid, propionic acid, and butyric acid exhibited a marked toxicity to Acidithiobacillus species. Of these organic acids, formic acid was the most toxic one as indicating that iron and sulfur oxidation almost were entirely inhibited at a concentration of 1.67 mM. In addition, it was found that A. ferrooxidans LX5 was more sensitive to glucose, starch, and citric acid than A. thiooxidans TS6, while the former seemed to be more acetic, propionic, and butyric acid resistant than the latter. In the selected 150 mg DOC L−1 of DOM derived from Sludge-H, the concentrations of formic acid and acetic acid were 8.94 mM and 2.09 mM, respectively, being a contributing factor causing 95% inhibition of iron oxidation and 70% inhibition of sulfur oxidation. To exploit specific heterotrophic microorganisms to eliminate these toxic organic compounds should be further studied.