1. The aim was to assess the extent to which the microbial biomass and activity, and community structure of fertilized upland grasslands are directly related to changes in soil N availability or indirectly related to individual plant species effects caused by changes in plant species composition and dominance. We investigated the short-term interactive effects of dominant plant species (Lolium perenne, Agrostis capillaris, Holcus lanatus and Festuca rubra) and nitrogen (N) amendment using an N-limited upland grassland soil. 2. In soils planted with different grass species, soil microbial biomass, and to some extent microbial activity, were determined by temporal changes in plant productivity. Variations in the way that individual plants influenced soil microbial biomass and activity were highly inconsistent over time, and largely independent of N-additions and differences in plant productivity. At the final sample date, those grass species which co-dominate the total plant biomass of intermediate fertility (H. lanatus) and semi-improved grasslands (A. capillaris and F. rubra) had a beneficial effect on the soil microbial biomass. In contrast, the dominant plant species of improved grasslands, L. perenne, had zero or a negative effect on soil microbial biomass. Two plant species (A. capillaris and H. lanatus) increased the proportion of fungi relative to bacteria in the soil microbial community, relative to the unplanted control soil and the other plant species. Lolium perenne and A. capillaris reduced the evenness of microbial PLFAs, suggesting negative effects of these plant species on the diversity of the soil microbial community. 3. The addition of N had no consistent effect on measures of soil microbial biomass or activity, but significantly altered the structure of the microbial community in favour of fungi. The lack of effects of N-addition on microbial biomass and activity were despite the finding that nitrogen addition reduced root biomass in all plant species and increased rhizosphere acidity. 4. The results suggest that in the short term, the abundance and activity of soil microorganisms in upland grasslands are regulated more by plant species traits than by a direct effect of nitrogen. These effects are likely to be related to variations amongst plant species in root exudation patterns and/or efficiency of nutrient acquisition. 5. Our study provides evidence that the functional characteristics of dominant plant species are important determinants of soil biological properties, and hence ecosystem functioning in temperate upland grasslands.