This investigation aimed to increase barley grain yield. In this respect, two field experiments were carried out at the Experimental Farm, Faculty of Agriculture (Saba Basha), Alexandria University, Alexandria, Egypt, in a split- split plot design with three replications during 2013/2014 and 2014/2015 growing seasons. Main plot treatments were sulphur rates (0, 200 and 400 kg/ha), however nitrogen fertilizer sources (urea, nitrate ammonium and ammonium sulphate) were allocated in sub-plots and biofertilizers inoculation (control, mycorrhizae and phosphorein inoculation) were distributed in sub- sub plots. The obtained results indicated that increasing sulphur application up to 400 kg/ha., significantly increased all studied yield, yield components and grain composition traits, i.e. number and weight of spikes/m2, number of spikelets and grains/spike, 1000-kernel weight, biological, straw and grain yield/ha, harvest index, grain protein, nitrogen, phosphorus and potassium content in the two studied seasons. Also, ammonium sulphate (as nitrogen source) produced the highest values of the previous trait. However, inoculated barley grains with phosphorein biofertilizers showed the highest of the studied traits except phosphorus grains content in the two seasons, where inoculation with mycorrhizae showed the highest grains phosphorus content (0.273 and 0.287%) in the two successive seasons. Sulphur application at 400 kg/ha., combined with ammonium sulphate or phosphorein inoculation interaction produced the highest values of all studied traits, except P and K contents in grains, meanwhile 400 kg S/ha application combined with mycorrhizal inoculation had the highest P and K content in two seasons. Ammonium sulphate X phosphorein inoculation interaction had the same trend in the two seasons. Regarding the three factors of interaction effects, sowing inoculated grains with phosphorein under 400 kg S/ha and ammonium sulphate application produced the highest values of the studied traits, except P and K grains content in both seasons. Conversely, any of two and three factors of interaction did not reach significant level effect on 1000- grains weight in the two seasons.