Ants are important ecosystem engineers and can be abundant in extensively managed grassland ecosystems. Different ant species create nests varying in structure and size, and tend to have a variety of feeding strategies. Differences in food imported to the nest and contrasting nesting behaviour may control soil microbial community structure in nest soil, with cascading effects on nutrient cycling, but this has not been tested in grassland ants. Soil and ants were sampled from nests of three ant species: two formicines; Lasius flavus (aphid farmer/scavenger, mound builder) and Formica lemani (scavenger/hunter, non-mound builder), and a myrmicine; Myrmica sabuleti (hunter/scavenger, non-mound builder), in an extensively grazed temperate grassland and compared to similar soils without ants. Microbial assemblages were determined using molecular approaches (terminal restriction length polymorphism and automated ribosomal intergenic spacer analysis). Both aboveground (vegetation diversity) and belowground (soil physico-chemical properties) components were measured to assess the potential of the different ant species to modify the environment. Stable isotope ratios (δ13C and δ15N) of ant tissues and nest soil organic matter confirmed differences in trophic distances. Significant changes in soil pH, moisture content, total C and total N, and in vegetation composition, demonstrated ant ecosystem engineering effects. In turn, nests of L. flavus, M. sabuleti and F. lemani had different microbial activities and harboured significantly different microbial assemblages (total bacteria, total fungi, ammonia-oxidising bacteria and nitrogen-fixing bacteria), but the diversity was similar. These findings suggest that grassland ants can control microbial assemblages via changes in physical and biological soil characteristics in their nests, and as such, different ant species harbour unique microbial assemblages in nests.