Sclerotinia sclerotiorumis an important plant pathogenic fungus. Biocontrol agents such as Bacillus spp. are promising candidates to manage the disease caused by this fungus. In this study, we identified 63 isolates of Bacillusspp. obtained from cucumber rhizosphere based on morphological, physiological and biochemical characteristics. The antagonistic activity of the isolates against the fungal pathogen was evaluated using dual culture method and the production of volatile metabolites. According to the obtained results, the isolates Ba17, Ba24, Ba14 and Ba29b showed the most inhibitory effects against the pathogen in dual culture method. Volatile metabolites of isolates Ba21, Ba29c, Ba10b, Ba17, Ba29b, Ba9c and Ba12a, reduced the mycelial growth of the pathogen by 87.14, 84.85, 83.42, 82.42, 79.14, 79.14 and 77.71%, respectively and had a significant difference compared with the control. In the greenhouse trails, isolates Ba17, Ba21, Ba10b, Ba29c and Ba14 decreased the disease index of Sclerotinia stem rot, significantly. The isolates Ba29c, Ba10b, Ba17, Ba29b, Ba24 were capable to show protease enzyme activity. Results also showed that isolates Ba29c, Ba10b and Ba21 were capable of producing surfactin. Molecular identification of isolates with antagonistic activities against the fungal pathogen by partial sequencing of 16S rDNA gene which amplificated with specific primers fD1 and rD1 was performed. Based on this procedure, isolates showed high homology withBacillus cereus, B. subtilis, B. licheniformis, B. endophyticusand Lysinibacillus spp..