Plant growth-promoting rhizobacteria (PGPR) are a group of beneficial soil bacteria that colonize the root surface and promote plant growth and health via multiple mechanisms. PGPRs are considered an eco-friendly alternative to hazardous chemical fertilizers. The use of PGPRs as biofertilizers is a biological approach toward the sustainable intensification of agriculture. Therefore, the current study investigated the possibility of two PGPBs, Azospirillum brasilense and Bacillus subtilis, to increase the bioavailability of Nitrogen (N) and phosphorus (P) in a calcareous soil fertilized with Ammonium Nitrate (AN) and single superphosphate (SSP) and its absorption by the barley (Hordeum vulgare L.). The pot experiment, a trial using calcareous soil, was conducted on December 1^st, 2022 for 60 days. The bacterial strains were mixed in the ratio (1:1) and tested in combination with four levels of Ammonium Nitrate and single super phosphate 0%, 50%, 75% and 100% of recommended dosage for barley (300 kg Fed^-1. Ammonium Nitrate and 150 kg Fed^-1. Single super phosphate). Treatments significantly increased the plant's dry weight, N and P absorption and bioavailable N and P in the soil compared to un-inoculated. Also, plants inoculated with bacteria had a significant impact on total amino acids as compared with un-inoculated plants. Accordingly, we can reduce Ammonium Nitrate and single super phosphate application by inoculation with these PGPR to have a major positive impact on barley growth and maintain environment and soil health.