Interactions between soil fauna and soil microorganisms are not fully recognized, especially in extreme environments, such as long-term metal-polluted soils. The purpose of the study was to assess how the presence of Lasius niger ants affected soil microbial characteristics in a long-term metal-polluted area (Upper Silesia in Poland). Paired soil samples were taken from bulk soil and from ant nests and analysed for a range of soil physicochemical properties, including metal content (zinc, cadmium, and lead). Microbial analysis included soil microbial activity (soil respiration rate), microbial biomass (substrate-induced respiration rate), and bacteria catabolic properties (Biolog® ECO plates). Soil collected from ant nests was drier and was characterized by a lower content of organic matter, carbon and nitrogen contents, and also lower metal content than bulk soil. Soil microbial respiration rate was positively related to soil pH (p = 0.01) and negatively to water-soluble metal content, integrated into TIws index (p = 0.01). Soil microbial biomass was negatively related to TIws index (p = 0.04). Neither soil microbial activity and biomass nor bacteria catabolic activity and diversity indices differed between bulk soil and ant nests. Taken together, ant activity reduced soil contamination by metals in a microscale which support microbial community activity and biomass but did not affect Biolog® culturable bacteria.