Combined application of organic and inorganic fertilizers can play an important role for increasing yield and quality of soybean (Glycine max L.). In order to study of effects of biofertilizers and nitrogen rates on yield, nodulation state and fatty acids composition of soybean, a factorial experiment was conducted based on randomized complete block design with three replications in 2012 and 2013. Factors were different rates of nitrogen fertilizer in four levels (without nitrogen and application 25, 50 and 75 kg urea/ ha) and seed inoculation with as biofertilizers in five levels (without inoculation, seed inoculation with Pseudomonas putida strain 41, P. putida strain 186, Azotobacter chroococcum strain 5 and Bradyrhizobium japonicum). The results showed that maximum of grain yield, plant height, number of filled pods and number of grains per plant were obtained from the highest level of nitrogen fertilizer (75 kg urea/ha) and Rh. inoculation. Furthermore, the highest rate of nitrogen usage (75 kg urea/ha) adversely inhibited nodulation of soybean. Number and dry weight of nodules/plant increased significantly with increasing nitrogen application rates up to 50 kg urea/ha. Seed inoculation with biofertilizers increased oil and protein contents. The maximum oil content was obtained by applying 50 kg urea/ha and seed inoculation with Bradyrhizobium. The saturated fatty acids (palmitic and stearic acids) declined in seed inoculation with Bradyrhizobium than the control, while it was vice versa in unsaturated fatty acids (linoleic, linoneic and oleic acids). Based on the results, it was concluded that application of suitable amounts of nitrogen fertilizer (i.e. between 50 and 75 kg urea/ha) as starter in seed inoculation with Bradyrhizobium japonicum can be recommended for profitable soybean production in the study area.