Plateau pikas cause changes in alpine meadow biodiversity and consequently impair ecosystem function through foraging, burrowing and defecation. However, existing studies often focus on a limited number of taxa, ignoring the fact that biodiversity loss occurs across many taxa and that the functional effects of any trophic group may depend on the diversity of other trophic groups. In this study, we analyzed the effects of disturbance by plateau pika on the diversity of organisms (plants, earthworms, ciliates, fungi and bacteria) and ecosystem multifunctionality (EMF) in different trophic groups. The results showed that: (1) disturbance by plateau pika significantly altered multi-trophic biodiversity and simultaneously affected EMF. (2) Plant species richness (25.52%) and functional richness (22.25%) were the most important factors driving EMF, followed by higher trophic level soil ciliates (18.35%) and earthworms (14.03%), and lower trophic level soil bacteria (9.88%), fungi, fungi, and bacteria (9.88%). Bacterial (9.88%) richness at lower trophic levels also drove EMF. (3) The relationship between biodiversity and EMF at each trophic level changed when the density of active burrows of plateau pikas was around 550 burrows/ha. At lower burrow densities (<530 burrows/ha), soil biodiversity had a greater effect on EMF, while at higher densities (>575 burrows/ha), plant diversity was more critical to EMF. (4) Disturbance by plateau pika had a negative effect on EMF, explaining 68% of the variation in EMF. This study reveals the important role of higher trophic level biodiversity in maintaining ecosystem function under the disturbance of plateau pika.