To assess the biocontrol potential of Nigrospora oryzae against Solanum rostratum, the effects of different medium components and fermentation conditions on the biomass of N. oryzae were investigated to determine the optimal medium composition and fermentation conditions. Subsequently, the pathogenicity of S. rostratum increased after artificial inoculation of S. rostratum with N. oryzae. Additionally, the safety of N. oryzae fermentation on seven crops were evaluated. The results revealed that the optimal shake flask culture ratio for N. oryzae was lactose:glycerol:yeast extract:(NH₄)₂SO₄ = 2:2:1:2. The optimal fermentation conditions were as follows: 15 mL of inoculum, 75 mL of fermentation liquid in a 150 mL shake flask, an initial pH of 5.0, a culture temperature of 20 °C, and 4 days of culture. The disease index of S. rostratum after spraying with N. oryzae fermentation agent was 60.00, indicating strong pathogenicity to S. rostratum. Safety evaluation revealed that N. oryzae exhibited no symptoms on Zea mays, S. melongena, Festuca arundinacea, Bromus inermis, or Medicago sativa but showed moderate susceptibility to Triticum aestivum and S. lycopersicum. This study represents the first exploration of the biocontrol potential of N. oryzae against S. rostratum. These findings suggest the potential of N. oryzae for development as a microbial herbicide targeting S. rostratum.