Objective To report the diagnosis and treatment processes of a fungal keratitis case caused by Lasiodiplodia theobromae (L. theobromae), and enhance the diagnosis and treatment experience on fungal keratitis caused by this rare pathogen. Methods Corneal scraping specimen from a patient with fungal keratitis was collec-ted. Gram-staining and fluorescence staining were conducted on specimen, followed with direct microscopic observation and isolation culture. The strain was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology and targeted DNA sequencing. Antimicrobial susceptibility testing was conducted. Literatures were summarized and clinical data on fungal keratitis caused by this pathogen were sorted out. Results Septal fungal hyphae could be seen on the corneal scraping specimen microscopically. The strain was identified as L. theobromae by both MALDI-TOF MS and targeted DNA sequencing after culture. Antimicrobial susceptibility testing (microdilution method) showed that the minimal inhibitory concentrations (MICs) of amphotericin B, vorionazole,itraconazole, posaconazole, fluconazole, 5-fluorocytosine, micafungin, caspofungin, and[LM]anidulafungin against this strain were 1, 0.25, > 8, 0.25, > 256, 8, 16, 2, and 0.25 μg/mL, respectively. Patient recovered well after antifungal treatment plan was adjusted according to antimicrobial susceptibility testing results. Conclusion L. theobromae is a rare pathogen that causes fungal keratitis. Laboratory tests provide rapid microscopic examination results, and take MALDI-TOF MS and targeted DNA sequencing identification techniques as effective means to detect rare pathogen. In vitro antimicrobial susceptibility testing result can provide reference for clinicians to correctly use antifungal agents for treatment of infection due to this pathogen.