Biochar as a soil amendment, increases soil fertility and better agricultural production. In addition, the utilization of microbial bio-fertilizers improves soil health and reduces the necessity of excessive chemical fertilizer application. In this regard, the present research was conducted to evaluate the effect of bio-inoculants and bagasse biochar on root colonization, grain nutrients uptake, growth, and yield of barley, and biological activities in a calcareous soil. An experiment was conducted as a factorial arrangement on a completely randomized design with three replications in a greenhouse condition for seven months. The treatments consisted of six bio-fertilizer levels including control, Rhizophagus intraradices, Funneliformis mosseae, Micrococcus yunnanensis, Rhizophagus intraradices + Micrococcus yunnanensis, Funneliformis mosseae + Micrococcus yunnanensis, and three bagasse biochar levels of 0, 1%, and 2% w/w. The application of 2% biochar significantly increased root colonization (32.3%), grain nutrients uptake of N, P, Zn and Cu (10%, 10%, 10%, and 4.3%, respectively), biological yield (5.08%), soil microbial respiration (SMR) (11.3%), and microbial biomass C (MBS) (12.7%) compared to that of B-0. Individual application of bio-inoculants and their combination with biochar increased root colonization, grain nutrients (Fe, Zn, Cu, Mn, P, and N) uptake, biological and grain yield, 1000-kernel weight, SMR, and MBS. Application of plant growth promoting microorganisms and bagasse biochar simultaneously makes positive effects on barley and microbial activities, which is a promising strategy.