Iron-sulfide minerals found in shale formations are stable under anaerobic conditions. However, in the presence of oxygen and water, acid-loving chemolithotrophic bacteria can transform the iron-sulfide minerals into a toxic solution of sulfuric acid and dissolved iron and minerals known as acid rock drainage (ARD). The objective of this study was to disrupt chemolithotrophic bacteria responsible for ARD using chemical treatments and to foster an environment favorable for competing microorganisms to attenuate the biologically induced ARD. Chemical treatments were injected into flow-through microcosms consisting of 501 g of pyrite-rich shale pieces inoculated with ARD bacteria. Three treatments were tested in the microcosms: (1) a sodium hydroxide-bleach mix, (2) a sodium lactate solution, and (3) a sodium lactate-soy infant formula mix. The effectiveness of the treatments was assessed by monitoring pH, dissolved iron, and other geochemical constituents in the discharge waters. The optimal treatment was a sequential injection of 1.5 g sodium hydroxide, followed by 0.75 g lactate and 1.5 g soy formula dissolved in 20 mL water. The pH of the discharge water rose to 6.0 within 10 days, dissolved iron concentrations dropped below 1 mg/L, the median alkalinity increased to 98 mg/L CaCO3, and sulfur-reducing and slime-producing bacteria populations were stimulated. The ARD attenuating benefits of this treatment were still evident after 231 days. Other treatments provided a number of ARD attenuating effects but were tempered by problems such as high phosphate concentrations, short longevity, or other shortcomings. The results of these laboratory microcosm experiments were promising for the attenuation of ARD. Additional investigations and careful selection of treatment methods will be needed for field application.