Sorghum bicoloris cultivated worldwide. Leaf spot of sorghum, which leads to leaf lesions and yield reduction, is a prevalent and serious disease in Guizhou Province, southwest China. In August 2021, new leaf spot symptoms were observed on sorghum leaves. In this study, traditional methods and modern molecular biology techniques were used to isolate and identify the pathogen. Sorghum inoculated with the isolate GY1021 resulted in reddish brown lesion that similar to symptoms observed in the field: the original isolate inoculated was reisolated and Koch’s postulates were fulfilled. Based on morphological features and phylogenetic analysis of the internal transcribed spacer (ITS) combined sequence with β-tubulin (TUB2) and translation elongation factor 1-α (TEF-1α) genes, the isolate was identified asFusarium thapsinum(Strain accession: GY 1021; GenBank Accession: ITS (ON882046),TEF-1α (OP096445), and β-TUB(OP096446)). Then, we studied the bioactivity of various natural products and microorganisms againstF. thapsinumusing the dual culture experiment. Carvacrol, 2-allylphenol, honokiol, and cinnamaldehyde showed excellent antifungal activity, with EC50values of 24.19, 7.18, 46.18, and 52.81 µg/mL, respectively. The bioactivity of six antagonistic bacteria was measured using a dual culture experiment and the mycelial growth rate method.Paenibacillus polymyxa,Bacillus amyloliquefaciensandBacillus velezensisdisplayed significant antifungal effects againstF. thapsinum. This study provides a theoretical basis for the green control of leaf spot of sorghum.