Abstract Root functional traits are known to influence soil properties that underpin ecosystem functioning. Yet few studies have explored how root traits simultaneously influence physical, chemical, and biological properties of soil, or how these responses are modified by common grassland perturbations that shape roots, such as defoliation and fertilisation. Here, we explored how root traits of a wide range of grassland plant species with contrasting resource acquisition strategies (i.e. conservative vs. exploitative strategy plant species) respond to defoliation and fertilisation individually and in combination, and examined cascading impacts on a range of soil abiotic and biotic properties that underpin ecosystem functioning. We found that the amplitude of the response of root traits to defoliation and fertilisation varied among plant species, in most cases independently of plant resource acquisition strategies. However, the direction of the root trait responses (increase or decrease) to perturbations was consistent across all plant species, with defoliation and fertilisation exerting opposing effects on root traits. Specific root length increased relative to non‐perturbed control in response to defoliation, while root biomass, root mass density, and root length density decreased. Fertilisation induced the opposite responses. We also found that both defoliation and fertilisation individually enhanced the role of root traits in regulating soil biotic and abiotic properties, especially soil aggregate stability. Synthesis: Our results indicate that defoliation and fertilisation, two common grassland perturbations, have contrasting impacts on root traits of grassland plant species, with direct and indirect short‐term consequences for a wide range of soil abiotic and biotic properties that underpin ecosystem functioning.