Soil biodiversity is essential for ecosystem multifunctionality (EMF). However, the relative contributions of soil biodiversity and biological interactions to EMF across multiple trophic levels, and the potential mechanisms remain uncertain under environmental variability, especially in fragile and highly heterogeneous alpine grasslands. Here, we investigated biodiversity (e.g., species diversity and community composition) and their interactions (quantified by network complexity) of individual soil trophic groups (bacteria, fungi, protists and nematodes) and multitrophic level with EMF (quantified using 18 variables) along altitude gradients (3400–4100 m) in alpine meadow on the eastern Qinghai-Tibet Plateau. The results showed that individual trophic biodiversity and network complexity had distinct patterns along altitude. At multitrophic level, although species diversity remained relatively stable, and community composition dissimilarity decreased, the network complexity increased with increasing altitude. EMF remained stable along altitude. No significant relationships between EMF and soil biodiversity were found at individual trophic and multitrophic levels. In contrast, EMF was positively correlated with multitrophic network complexity, and this positive association was strengthened with increasing altitude, although it was not correlated with network complexity of individual trophic groups. Further pathways analysis indicated that multitrophic biodiversity had no significant effect on multitrophic network complexity and EMF. However, environmental conditions (low temperature and high soil moisture, which are prevalent at higher altitudes) enhanced multitrophic network complexity, with high soil moisture in particular facilitating bacterial-fungal synergism, thereby positively driving EMF along altitude gradients. This study highlighted the crucial role of soil multitrophic interactions in supporting EMF in alpine grasslands. These results could offer insight into soil biodiversity-EMF relationship and theoretical basis for conservation and management of alpine grasslands.