Diversity and composition of plants and soil biota are critical determinants of fundamental ecosystem functions. Although the compositional responses of organisms across trophic levels to global change drivers and ecosystem management strategies have been well documented, their intra-annual variations and main drivers remain largely unknown. We hypothesized that the intra-annual dynamics of each biotic group would be driven by distinct factors: light availability for plants, temperature and plant growth for soil microbe, and soil nutrients for nematodes. We investigated the taxonomic diversity and community composition of plants, soil bacteria, fungi, and nematodes across five months in a grassland experiment following eight years of factorial treatments involving nitrogen (N) addition and mowing. We found significant intra-annual variations in the community composition of plants, soil bacteria, fungi, and nematodes. However, the primary drivers of these variations differed among biotic groups. Specifically, light availability, plant above-ground biomass, temperature, and soil nutrient dynamics played dominant roles in shaping plant, bacterial, fungal, and nematode communities, respectively. Neither N addition nor mowing significantly altered the intra-annual variation pattern of biotic groups, except for plants. Nitrogen addition significantly affected the intra-annual stability of community compositions across all biotic groups and disrupted their inherent correlations. By elucidating the key drivers of seasonal variations in the community composition of plants, bacteria, fungi, and nematodes, this study advances our mechanistic understanding of intra-annual dynamics in grassland ecosystems under nutrient enrichment and provides insights for biodiversity conservation in a changing world.