Background: Alfalfa (Medicago sativa L.) is one of the most productive plant in terms of nitrogen fixation and the suitable amount of protein and carotene has a significant impact on the nutritional quality of alfalfa. Methods: This combined experiment was conducted as factorial based on a randomized complete block design with three replications during two consecutive years (2019-2020) under the climatic conditions of Jiroft area (Kerman province, Iran). The studied factors included alfalfa age (one, three and five-years old) and bio-fertilizers (Control, Potash-Barvar (KSB), Phosphate Barvar II (PSB), Potash-Barvar + Phosphate Barvar II) (KSB+PSB). The biofertilizers were used along with irrigation water. The studied physiological traits included nitrogen, phosphorus, crude protein, soluble sugars, malondialdehyde (MDA) and sodium amount. Result: The highest amount of crude protein, nitrogen, phosphorus and soluble sugars was observed in three-years old fields under the treatment of bio-fertilizers Potash Barvar + Phosphate Barvar II (KSB+PSB) (37.78%, 9.79%, 0.51% and 324.75% respectively). Unlike other treatments, the highest amount of sodium (0.95%) and malondialdehyde (MDA) (83.99 μM g-1FW) was measured for alfalfa in treatment one-years old field under no-fertilizers (control), while the lowest amount of sodium (0.49%) and malondialdehyde (MDA) (55.94 μM g-1FW) was observed in treatment five-years old field under bio-fertilizers Potash Barvar + Phosphate Barvar II (KSB+PSB) application.