A field study was conducted to evaluate the efficacy of biocontrol agents on controlling root rot on Nigella sativa L. plants in two successive seasons 2018/2019 and 2019/2020 in the Experimental Farm of South Tahrir Horticulture Research Station, at Ali Mubarak farm, El- Behira Governorate, Horticulture Research Institute (HRI), Agriculture Research Center (ARC), Egypt. Ten isolates of Bacillus spp. and Streptomyces spp. (5 Bacillus and 5 Streptomyces) were evaluated for their effectiveness against Rhizoctonia solani. B. subtilis, B. licheniformis and S. rochei were used in vivo in two forms powder and suspension to suppress R. solani root rot. The results showed that Streptomyces rochei (OP164572), Bacillus subtilis (MT110640) and Bacillus licheniformis (OP164574) gave the highest reduction on growth of R. solani (55.47%, 60.17% and 59.53%) respectively, compared with control. Correspondingly, when biologically treated plants were compared to untreated control plants, physiological and biochemical indicators were likewise significantly elevated. Under field condition, our results revealed that used suspension form was more effective than powder form for management of root rot disease in Nigella sativa L., plants. Results showed that, when compared to untreated plants, all biocontrol agents decreased the frequency of root rot and improved vegetative growth, seed yield, volatile oil production, and fixed oil. The treatment with B. subtilis suspension produced the highest percentage of survived plants (98% vs. 14% in the control), as well as the highest growth, seed yield, volatile oil, and fixed oil content followed by S. rochei suspension, while treated by B. licheniformis was the less effective. The effect of bioagenst against R. solani was examined under fixed camera light microscope. B. subtilis, B. licheniformis, and S. rochei suspensions can be recommended as fungicides alternatives for controlling black cumin root rot disease in greenhouse and field environments.