Alfalfa is a widely distributed forage legume whose leaves are high in protein content and whose stems are suitable for bioethanol production. However, alfalfa forage digestibility, quality and yield may vary under future climate change scenarios. This legume can establish double symbiosis with nitrogen-fixing bacteria and arbuscular mycorrhizal fungi (AMF). The presence of AMF can modify the evolution of biomass production and partitioning during the vegetative growth of alfalfa. We hypothesised that mycorrhizal symbiosis may change the quantity and/or quality of carbohydrates and lignin in leaves and/or stems of alfalfa, with these changes being dependent on the atmospheric CO2 concentration at which plants are grown. Results showed that mycorrhizal alfalfa plants exposed to elevated CO2 had improved leaf, stem and root biomass, enhanced amount of hemicellulose and decreased concentration of lignin in cell walls of leaves as well as increased levels of glucose and fructose in stems compared with non-mycorrhizal alfalfa. These results indicated improved forage quality (leaves) and enhanced potential for bioethanol conversion (stems) in mycorrhizal alfalfa cultivated under elevated CO2. Moreover, the potential of stems for producing CH4 reinforced their suitability for the conversion of biomass into bioethanol.