Buffalobur (Solanum rostratum Dunal) () is an invasive weed in China, identifying its pathogens is crucial for developing effective biological control measures. In this study, leaf samples from S. rostratum showing typical disease symptoms were collected in Liaoning and Jilin provinces, China. The isolated fungal pathogens were identified based on their morphological characteristics and by using molecular biology techniques. Pathogenicity was assessed by artificially inoculating spore suspensions from the fungal pathogen onto the seeds, isolated leaves, and plants of S. rostratum. The safety of the fungal pathogens for eight plant species was also evaluated. We then identified the following five fungal pathogens causing disease in S. rostratum in Liaoning and Jilin provinces: Alternaria alternata, Epicoccum sorghinum, Fusarium equiseti, Curvularia hawaiiensis, and Nigrospora oryzae. These fungal pathogens exhibited pathogenicity, with N. oryzae exhibiting the strongest pathogenicity and highest safety. N. oryzae demonstrated the highest inhibition rate against the radicle germination length of S. rostratum and showed robust pathogenicity towards both isolated leaves and plants. Notably, despite inducing mild reactions in corn, sorghum, rice, and tomatoes, N. oryzae did not impose any detrimental effects on the growth of these plants.