This study had the objective of evaluating the effect of biofertilizers on the performance of soybean plants under different planting spacing. It was conducted during the summer seasons of 2018 and 2019at one site at Demonstration Farm of Damazin Agricultural Research Station, Sudan. The different plant spacing designated as D1, D2, and D3 (5, 10, and 15 cm between plants) under three types of bio-fertilizers as Rhizobacteria (B1), Azotobacter (B2), Bacillus (B3) and control (B0). The experiment was laid out in Randomized Complete Block design (RCBD) with three replications as split-plots trail. The result revealed that the improvement due to the application of fertilizers with wider, spacing D3 stimulated plant growth and caused an increase in dry matter accumulation and high leaf area and gave the higher number of pods and seeds/pod, particularly at inoculation with B1 and B2 bacteria. While sowing seeds inoculated with B1or B2 under closer spacing resulted in higher seed yield per unit area. To achieve optimum results in soybean variety Sudan 1, the crop may be sown in plant spacing of 10cm with inoculation seeds with B1or B2 to achieve a high plant population per unit area to obtain higher seed yield.