Research was conducted to assess the response of microbial communities in a soil with a long history of glyphosate-based herbicides to a secondary imposed perturbation (dry–rewetting event). Both perturbations could increase their frequency under current agricultural practices and climate change. The results of this study demonstrate that acute exposure to a glyphosate-based herbicide does not have a conditioning effect on the response of microbial communities to the dry–rewetting event.