Forest pest damage is expected to increase with global change. Tree diversity could mitigate this impact, but unambiguous demonstration of the diversity–resistance relationship is lacking in semi-natural mature forests. We used a network of 208 forest plots sampled along two orthogonal gradients of increasing tree species richness and latitudes to assess total tree defoliation in Europe. We found a positive relationship between tree species richness and resistance to insect herbivores: overall damage to broadleaved species significantly decreased with the number of tree species in mature forests. This pattern of associational resistance was frequently observed across tree species and countries, irrespective of their climate. These findings confirm the greater potential of mixed forests to face future biotic disturbances in a changing world.