Abstract: Tobacco black shank caused by Phytophthora nicotianae is an important and destructive soil-borne disease affecting tobacco production in China. Biofumigation is a method to suppress or kill harmful organisms in the soil using volatile bioactive substances produced by plants, such as those of Cruciferae or Compositae, during decomposition. Brassica species are important biological fumigants. ‘Dilong 1’ (DL1) mustard was cultivated by our collaborator at Nanjing Agricultural University as Brassica juncea variety with high glucosinolates content, high biomass, high adaptability, and good fumigation effects. To explore a safe and environmentally friendly green method for the prevention and control of tobacco black shank disease, the inhibitory effect of fumigation with DL1 mustard on P. nicotianae was analyzed in this study through a series of petri dish inocubation experiments. The mycelial growth rate method was used to determine the effect of DL1 fumigation on mycelial growth of P. nicotiana. The fumigation effects on sporangium formation, zoospore release, spore germination, germ tube elongation, and oospore formation of P. nicotiana were also determined. The effects of DL1 fumigation on cell membrane permeability of P. nicotiana were determined by changes in electrical conductivity and malondialdehyde content. The change in the pathogenicity of P. nicotiana after fumigation was determined using the in vitro leaf method. The results showed that fumigation of fresh matter of DL1 mustard could significantly inhibit the mycelial growth of P. nicotiana. The EC₅₀ (concentration for 50% of maximal effect) value was 0.362 g, EC₇₅ (concentration for 75% of maximal effect) value was 0.499 g, and minimum inhibitory dose (MIC) was 0.500 g. The inhibitory effect was dose-dependent on the amount of fresh material in DL1 mustard. The amount of 0.3 g fresh DL1 mustard significantly inhibited sporangium formation and zoospore release of P. nicotiana, and the amount of 0.5 g could inhibit sporangium formation and zoospore release of P. nicotiana by 72.8% and 86.6%, respectively. When the amount of fresh DL1 mustard was increased to 0.7 g, sporangium formation of P. nicotiana was completely inhibited. The amount of 0.3 g and 0.4 g of fresh DL1 mustard could completely inhibit zoospore germination and germ tube elongation of P. nicotiana, respectively. Fumigation of DL1 could reduce the formation of oospores of P. nicotiana in a dose-dependent manner, and 0.7 g fresh DL1 mustard could completely inhibit the oospore formation of P. nicotianae. After fumigation, the permeability of the mycelium membrane of P. nicotianae increased, and membrane lipid peroxidation was induced. After 150 min of fumigation, the electrical conductivity of P. nicotiana was 2.2 times that of the control, and the malondialdehyde content was 8.0 times that of the control. The fumigation treatment weakened the pathogenicity of P. nicotianae, and the area of the disease spot decreased by 88.9% compared to that of the control. In this study, fumigation of DL1 mustard on the growth and development of P. nicotianae, physiological and biochemical characteristics, and pathogenicity changes indicated that it had a good inhibition and killing effect on P. nicotianae, and the fumigation effect had a dose-dependent effect on the amount of fresh DL1 mustard. This provided a theoretical basis for the use of DL1 mustard to control tobacco black shanks and other soil-borne diseases in production.