When reduced sulfidic parent sediments are oxidized, they become acid sulfate soils and discharge metal laden acidic solutions that can damage the environment, infrastructure, and human health. Consequently, methods to mitigate the effect of acid sulfate soils are a priority in affected areas. In this study, acid sulfate soil core samples, consisting of a natural network of preferential-flow soil macropores with defined macropore surfaces and inner cores of denser clay, were characterized and subjected to treatments with calcium carbonate and peat suspensions, or combinations thereof. The effects on the geochemistry and microbial communities were examined on both macropore surfaces and in inner cores. Although transport of treatment substances into the inner cores was demonstrated, no substantial effects were found on the geochemistry and microbial community that consisted of bacterial taxa commonly identified in acid mine drainage. In contrast, positive treatment effects were clearly detected on macropore surfaces and the most promising mitigation effects were detected for treatments combining calcium carbonate and peat suspensions. These treatments increased the pH of the macropore surfaces, added an electron donor in the form of peat, and significantly decreased the relative abundance of acidophilic bacterial populations while shifting the microbial community towards species typically growing at circumneutral pH values. These new environmental conditions were favorable for iron reduction that resulted in a positive effect on permeate quality. The study presents novel data regarding the important differences between acid sulfate soil macropore surfaces and inner cores, as well as their diverse biogeochemical characteristics. It further establishes that the major oxidation-reduction processes occur at the macropore surfaces, and that the combination treatment was the most effective at mitigating the negative environmental effects.