Abstract Ecotypic differences in plant growth and anti-herbivore defence phenotypes are determined by the complex interactions between the abiotic and the biotic environment. Root-associated microbes (RAMs) are pervasive in nature, vary over climatic gradients and have been shown to influence the expression of multiple plant functional traits related to biomass accumulation and biotic interactions. We addressed how variation in climatic conditions between lowland and subalpine habitats in the Alps and RAMs can independently or interactively affect plant growth and anti-herbivore defence trait expression. To address the contribution of climate and RAMs on growth and chemical defences of high- and low-elevation Plantago major ecotypes, we performed a full-factorial reciprocal transplant field experiment at two elevations. We coupled it with plant functional trait measurements and metabolomics analyses. We found that local growing climatic conditions mostly influenced how the ecotypes grew, but we also found that the high- and low-elevation ecotypes improved biomass accumulation if in the presence of their own-elevation RAMs. We also found that while chemical defence expression was affected by climate, they were also more highly expressed when plants were inoculated with low-elevation RAMs. Synthesis. Our research demonstrated that root-associated microbes (RAMs) from contrasted elevations impact how plants grow or synthesize toxic secondary metabolites. At low elevation, where biotic interactions are stronger, RAMs enhance plant biomass accumulation and the production of toxic secondary metabolites.