Alfalfa (Medicago sativa L.) is cultivated in arid and semi-arid regions where salinity is one of the main limiting factors for its production. Thus, this experiment was conducted to evaluate the efficacy of arbuscular mycorrhizal fungus (AMF), Glomus mosseae, alfalfa rhizobia Sinorhizobium meliloti (R) seed inoculation in the development of salinity tolerance of different alfalfa cultivars (Rehnani, Pioneer and Bami) under a variety of salinity levels. The results revealed that under non-stress condition, root mycorrhizal infection, nodulation (the number and weight of nodules per plant), potassium (K), calcium (Ca), phosphorus (P), zinc (Zn), copper (Cu) and magnesium (Mg) contents of the root and shoot, the value of the K/ Na ratio, protein [calculated from the nitrogen (N) content] and proline contents of the shoot and the alfalfa yield were found to be the highest while Na contents of the root and shoot were seen to be the lowest when seeds were double inoculated followed by mycorrhizae, rhizobium and control treatments, respectively. Similarly, under salinity condition, the greatest amounts of mycorrhizal infection, nodulation, root and shoot P contents, the value of K/Na ratio, the shoot proline content and the root Ca content were enhanced with the least amount of leaf Na content related to the cases of seeds which were double inoculated, followed by mycorrhizae, rhizobium and control treatments respectively. The results suggested that inoculation of alfalfa seed with AMF or R, especially double inoculation, causes a considerable increase in alfalfa yield under both saline and non-saline conditions by increasing colonization, nodulation and nutrient uptake.