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Zinnia elegans Jacq. is an important, globally cultivated ornamental plant. In August 2021, a leaf spot disease was observed in zinnia in Shibing County, Guizhou, China, with an incidence of approximately 60%. Pathogens were isolated and purified from the infected leaves by tissue isolation, and pathogen strain BRJ2 was confirmed as the pathogen causing the leaf spot. Based on morphology and ITS, TEF-1α, and TUB2 sequence analyses, the pathogen was identified as Nigrospora musae (McLennan and Hoëtte). The mycelial growth rate method was used to determine the in vitro toxicity of five fungicides to the pathogen. The results showed that 10% difenoconazole provided the strongest inhibitory effect on N. musae, with a concentration for 50% of maximal effect (EC50) of 0.0658 mg/L; 75% trifloxystrobin·tebuconazole had the second greatest effect, with an EC50 of 0.1802 mg/L. This study provides the first report that N. musae caused leaf spot disease in Z. elegans and provides important guidance for the effective prevention and control of this disease in Guizhou.