Seed irradiation during pre-sowing processes is one of the most effective methods to increase plant growth, yield components and its chemical composition. A three factor split-split plot, factorial experiment was conducted on barley to study seed irradiation, biofertilization with N2 fixing bacteria (NFB), P-dissolving bacteria (PDB) and N fertilization in the Agricultural Research Station of El-Zagazig University at El-Khattara region, El-Sharkia Governorate, Egypt during 2009/2010 season. Main plots were assigned to gamma irradiation: non-radiated, 20 and 40 Gy gamma radiations. Sub-plots were assigned to N: 0, 119, 238 and298 kg N ha-1. Sub–sub plots were assigned to biofertilization: none, (Azospirillum lipoferum + Bacillus polymxa “NFB1"), (Azospirillium braselence + Azotobacter chroccocum “NFB2"+Bacillus megatherium “PDB") and NFB1 + NFB2 + PDB. The NFB1 was given in a commercial product of “Cerealin" while (NFB2 + PDB) was in a product of Microbin. Barley yield and yield components as well as N, P and K content and uptake increased by gamma irradiation, N rates and biofertilization singly or combined. Number of spike per m2, grain weight per spike, yields of: grains + straw, grains and straw increased also, highest response occurred by N238 + (Cerealin + Microbin) with 40Gy gamma ray. The highest straw yield was observed due to N179 + (Cerealin + Microbin) with 40Gy gamma ray. Treatment of N179 + Cerealin with 40Gy gamma ray gave highest N-uptake in straw, as well as P-uptake in grains and straw. Protein content and protein yield increased by treatments especially with increasing N rate up to N298 + Microbin + 20Gy and gave highest protein content. Nitrogen use efficiency (NHI) was increased with increasing N rates up to N238 followed by decrease at N298.