This study aimed to investigate the impact of two isolates of Bacillus simplex (PHYB1 and PHYB9) for controlling the root rot disease of black cumin (Nigella sativa) caused by Fusarium camptoceras (PHYF1) under greenhouse and field conditions at Assiut Governorate, Egypt. The highest reduction percentage of infection (22.5%) was recorded by the treatment of PHYB1 as suspension than the control (60%). Both isolates of Bacillus spp. provided the root and foliar dry weight under greenhouse and seed production in the field. The results showed that the isolate PHYB1 as a formulation gave the highest impact in the root dry weight (0.28 g/plant), followed by PHYB9 (0.22 g/plant), with insignificant difference between them. PHYB9 suspension also provided the highest seed production (27.97 g/plant), whereas PHYB9 as a formulation gave the lowest (24.08 g/plant). Studies on the interaction between Bacillus spp. on F. camptoceras by scanning electron microscope (SEM) revealed that both caused a complete mycoparasitism on the fungal growth. The bacterial growth was seen to adhere and colonize the hyphae, resulting in hyphal tissue maceration. Therefore, the use of both isolates of Bacillus spp. to control root rot disease of black cumin under greenhouse and field conditions can be recommended.