The purpose of this paper is to study multi-derivations of Poisson algebras. First, we show that the space of multi-derivations of a Lie algebra 𝔤 with the Nijenhuis–Richardson bracket is a differential graded Lie algebra. Then we introduce the notion of a multi-derivation of a Poisson algebra, and show that the space of multi-derivations of a Poisson algebra with the Nijenhuis–Richardson bracket is also a graded Lie algebra. Finally, we study multi-derivations of three concrete Poisson algebras coming from linear Poisson manifolds, symplectic manifolds and Poisson manifolds with vanishing first cohomology groups. We establish the relationship between multi-derivations of a Lie algebra 𝔤 and multi-derivations of the linear Poisson manifold 𝔤∗, and show that a two-derivation of a connected symplectic manifold (M,ω) is λπ, where π is the Poisson bivector field induced by the symplectic structure ω and λ is a real number. We also prove that a two-derivation of a connected Poisson manifold (M,π) with trivial Poisson cohomology group H1(M,π) is fπ, where f is a Casimir function.