Egypt faces challenge in supplementing animal feed requirements which add huge pressure on the budget and foreign currency reserves annually leading to the importance of finding alternative solutions. The sprouted barley is considered one of these recent alternatives to animal feed which faces challenges in controlling the rate of seed germination due to the growth of fungi that consume the oxygen necessary for germination in addition to aflatoxins formation and its harmful effect on animal health. Biofertilizers plant growth promoting bacteria PGPB is considered a practice and safe solution. In this work, five tomato rhizobacterial strains were isolated and identified using 16S rRNA gene and were closely related to Bacillus amyloliquefaciens, Peribacillus frigoritolerans, Pseudomonas flourescens, Bacillus pumilus, and Paenibacillus uliginis, respectively. We reported here that most of these five isolates exhibited multiple PGP characteristics (PGPC), including the production of ACC deaminase, Indole-Acetic Acid (IAA), chelating siderophores and phosphate solubilization. Bacillus amyloliquefaciens BMG150 isolate exhibited the highest values for all the PGPC except siderophores production (1457 nmol, 37.4 µg/ ml, and 3.7 mg/ml, respectively). We also scanned the presence/ absence of the non-ribosomal peptide gene clusters in the five isolates as an important PGPC using bioinformatics tools and NRPs degenerate primers. All five isolates showed the presence of NRPs gene clusters with the superiority of NRPs number for the strain Bacillus amyloliquefaciens BMG150 (surfactin, fengycin or plipastatin, iturin and bacillibactin siderophore). According to these results, we used this latter strain, Pseudomonas flourescens PMG01 separately and a formula of the other three isolated strains as biofertilizers in sprouted barley cultivation which proved their efficiency in promoting their growth characteristics and reduced fungal growth which reflected on protein pattern.