Barley (Hordeum vulgare L.) is cultivated globally under a wide range of climatic conditions and is subjected to chilling and freezing stresses under temperate and cold climatic conditions. As a mycorrhizal crop, barley may benefit from this association for increasing cold resistance. In order to investigate the effects of inoculation with arbuscular mycorrhizal fungi (AMF) on cold-stress resistance in barley plants, one winter and one spring cultivar were grown under control (25°C day, 17°C night) and low, non-freezing (LT: 5°C day, 3°C night) temperatures for 3 weeks in the absence (−AMF) or presence (+AMF) of two species of AMF, Glomus versiforme and Rhizophagus irregularis. In addition, the influence of LT (as an acclimation treatment) was studied on plant survival after a 2-day exposure to freezing temperature (FT: −5°C in dark). Biomass production, membrane integrity and survival rate of plants indicated that the winter cultivar was more tolerant than the spring cultivar. Inoculation with AMF resulted in improved growth, photosynthesis, osmotic and water homeostasis, and potassium uptake under both control and LT conditions, whereas the effect on membrane integrity, antioxidative defence and phenolics metabolism was mainly observed in LT plants. AMF inoculation substituted partially or completely for acclimation treatment and increased the survival rate of FT plants, with the highest survival achieved in a combination of AMF and LT. Mycorrhizal responsiveness was higher in LT plants. Despite the lower AMF colonisation, G. versiforme was often more effective than R. irregularis for the alleviation of low temperature stress in both cultivars, whereas R. irregularis was more effective in increasing the survival rate. Our data suggest that the right combination of fungus species and host-plant cultivar is important for successful utilisation of AMF under cold conditions.