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Under salinity conditions, growth and productivity of grain crops decrease, leading to inhibition and limited absorption of water and elements necessary for plant growth, osmotic imbalance, ionic stress, and oxidative stress. Microorganisms in bio-fertilizers have several mechanisms to provide benefits to crop plants and reduce the harmful effect of salinity. They can be effective in dissolving phosphate, fixing nitrogen, promoting plant growth, and can have a combination of all these qualities. During two successful agricultural seasons, two field experiments were conducted to evaluate the effect of bio-fertilizer applications, including phosphate solubilizing bacteria (PSB), nitrogen fixation bacteria and a mix of phosphate-solubilizing bacteria and nitrogen fixation bacteria with three rates, 50, 75 and 100% NPK, of the recommended dose of minimal fertilizer on agronomic traits, yield and nutrient uptake of barley (Hordeum vulgare) under saline condition in Village 13, Farafra Oasis, New Valley Governorate, Egypt. The results showed that the application of Microbein + 75% NPK recorded the highest values of plant height, spike length, number of spikes/m2, grain yield (Mg ha−1), straw yield (Mg ha−1), biological yield (Mg ha−1), protein content %, nitrogen (N), phosphorus (P), potassium (K) uptakes in grain and straw (kg ha−1), available nitrogen (mg/kg soil), available phosphorus (mg/kg soil), total microbial count of soil, antioxidant activity of soil (AOA), dehydrogenase, nitrogen fixers, and PSB counts. The application of bio-fertilizers led to an increase in plant tolerance to salt stress, plant growth, grain yield, and straw yield, in addition to the application of the bio-fertilizers, which resulted in a 25% saving in the cost of mineral fertilizers used in barley production.