Application of biological fertilitzers to eliminate nutrient deficiency of nutrient elements i.e. Fe and Zn is one of the most important purposes of soil fertility. Microorganisms which are used as biofertilizer, have several mechanisms such as siderophore production to increase nutrient uptake. Siderophores are chemical ligands with high affinity to bind with iron. The purpose of this experiment was to investigate the effect of individual and simultaneous inoculation of Sinorhizobium meliloti and Piriformospora indica on zinc and iron uptake by alfalfa ( Medicago sativa ) in response to different levels of Fe (2.5, 50 µM) and Zn (0, 1, 2 µM). Therefore, a completely randomized block design with 3 replications was carried in greenhouse Soilless Culture Research Center of Isfahan University of Technology. The results showed that individual and simultaneous inoculation of the studied microrganisms specially individual inoculation of S. meliloti caused increasing (p 0.05) root and shoot dry weigth of alfalfa compared to the control. Individual inoculation of P.indica and simultaneous inoculation of the fungus and bacterium at the Zn-free and 2.5 mM Fe trearments increased the shoot Zn concentration by 44% and 58% respectively compared with the control plants, but in such conditions the effect of Sinorhizobium meliloti on this parameter was not significant. The results indicoted the positive effect of S. meliloti inoculation on root and shoot Fe concentration. Individual inoculation of fungus and bacterium had significant effect on shoot and root phosphorus concentration. Also, P.indica was more effective on the shoot nitrogen content than the other treatments. Results showed that individual inoculation of P.indica and simultaneous inoculation of this fungus with S. meliloti increased the activity of guaiacol peroxidase (GPX) at the Zn- and Fe-free treatments. The microbial inoculation had no significant effect on ascorbate peroxidase (APX) activity. In general, with respect to the positive effects of individual and simultaneous inoculation of the studied microorganisms on root and shoot growth of alfalfa, these microorganisms can be introduced as biofertilizers. Although further research is needed. Keywords : Biological fertilitzers, Siderophore, Sinorhizobium meliloti , Piriformospora indica