Our aim was to study the effects of glyphosate, tilling practice and cultivation history on mycorrhizal colonization and growth of target (weeds) and non-target (crops) plants. Glyphosate, the world's most widely used pesticide, inhibits an enzyme found in plants but also in microbes. We examined the effects of glyphosate treatment applied in the preceding fall on growth of a perennial weed, Elymus repens (target plant) and a forage grass, Festuca pratensis (non-target plant) and their arbuscular mycorrhizal fungal (AMF) root colonization in a field pot experiment. Non-target plants were sown in the following spring. Furthermore, we tested if glyphosate effects depend on tillage or soil properties modulated by long cultivation history of endophyte symbiotic grass (E+ grass). AMF root colonization, plant establishment and growth, glyphosate residues in plants, and soil chemistry were measured. Glyphosate reduced the mycorrhizal colonization and growth of both target and non-target grasses. The magnitude of reduction depended on tillage and soil properties due to cultivation history of E+ grass. We detected glyphosate residues in weeds and crop plants in the growing season following the glyphosate treatment. Residues were higher in plants growing in no-till pots compared to conspecifics in tilled pots. These results demonstrate negative effects of glyphosate on non-target organisms in agricultural environments and grassland ecosystems.