The yellow-meadow ant (Lasius flavus) is a common mound-building species of central European grasslands. Lasius flavus activity has been shown to change the amounts of plant nutrients in their mound soil substrate. It is unknown how the maturity of such mounds influences nutrient status and affects plant growth. We quantified bulk soil and extractable nutrient contents of three mound development stages (young, middle-aged and fully-developed) in comparison to the surrounding soil (control), and in a bioassay using species of three plant functional types (grass, non nitrogen (N)-fixing forb, N-fixing forb) linked these to plant growth and chemistry. Our results showed that development stage was an important predictor of a mound’s nutritional status. Lasius flavus activity results in decreases in bulk soil organic carbon, total nitrogen, sulfur, extractable manganese and zinc, and increases in bulk soil clay, pH, base cations, iron, phosphorous, boron, extractable calcium and potassium. The relative amounts of accumulated biomass, the proportion allocated to shoots or roots, and the N content of these tissues all differed among plant species. The N-fixing forb gained the highest total biomass and had the greatest allocation to shoots on soils of fully-developed mounds. The grass responded to all mound development stages with increased root biomass, whereas no effect was detected for the non N-fixing forb. The root N contents of the grass and the non N-fixing forb decreased with mound development stage. The findings highlight the importance of established L. flavus mounds as discrete micro-habitats in grassland ecosystems adding to biogeochemical heterogeneity.