Abstract: Fourteen Bacillus subtilis isolates (B1 to B14) obtained from different Egyptian sites had different antagonistic and inhibitory effect against six fungal isolates belonging to four different genera, Rhizoctonia solani, Helminthosporium sp., Alternaria sp. and Fusarium oxysporum. Random Amplified Polymorphic DNA (RAPD), differential display technique and partial sequencing of 16S rRNA gene were used for characterization of isolates. While RAPD technology, using 4 different random 20-mer primers, revealed different levels of molecular variation among twelve out of the fourteen isolates tested, the remaining two isolates B3 and B5, demonstrated identical profiles. Because of the high degree of homology (93%) between isolates B4 and B7 and the no variation observed between isolates B3 and B5, the differential display technique was used to test the reliability of these results. Isolate B7 was selected for batch cultivation in bioreactor, the biomass achieved was 3.2 g L-1. Inhibitory activity of supernatant was increased near the end of the stationary phase. The activity reached its highest value of 1.7 cm in death phase.