Fusarium wilt of tomato is one of the most prevalent and economically important diseases of tomato worldwide especially in tropical regions. The aims of the present study were to isolate and characterize Bacillus bacteria from tomato rhizospheric soil of various regions in Iran and determine the isolates that exhibit high levels of antagonistic efficiency against tomato Fusarium wilt pathogen, Fusarium oxysporum f. sp. lycopersici (Fol) and growth promotion activity. In this study, 303 Bacillus isolates were obtained from tomato rhizospheric soil. Dual culture and volatile metabolite tests were used to screen for antagonism of Bacillus isolates against Fol. Among them, 20 isolates were found to inhibit pathogen growth by 67.77% and 33.33% in dual culture and volatile metabolite tests, respectively. Based on the results of physiological tests and 16S rRNA and gyrA gene sequence analysis of 20 effective isolates, 11, seven and two isolates were identified as B. subtilis, B. velezensis and B. cereus, respectively. The results of greenhouse assessment showed that KR1-2, KR2-7 and A2-9 isolates which were characterized as Bacillus subtilis, reduced the disease index to 16.67% and promoted the plant growth by 80%. These isolates may serve as potential promising biocontrol agents against Fusarium wilt of tomato.