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To evaluate in vitro susceptibility to topical antifungal medications, as measured by minimum inhibitory concentration (MIC) and 50% inhibitory concentration (IC50%), of fungal isolates from horses with ulcerative keratomycosis in Florida; to compare results with those of other studies to identify differences in susceptibility patterns among fungi isolated from horses in different geographic regions; and to note indications of fungal resistance to drugs tested in other studies.Corneal fungal cultures from client-owned horses from Florida with ulcerative keratomycosis (n = 22).Fungal cultures were plated on Emmons modified Sabouraud dextrose agar and mycobiotic agar, examined weekly for growth, and kept for a total of 30 days. In vitro MIC and IC50% of fluconazole, itraconazole, ketoconazole, miconazole, and natamycin were measured for each fungal isolate.Aspergillus (n = 9; 41%), Fusarium (7; 32%), Penicillium (2; 9%), Cylindrocarpon (1; 4%), Scytalidium (1; 4%), and Torulopsis (1; 4%) spp and an unidentified yeast (1; 4%) were isolated. Fungi were most susceptible to antifungal drugs in the following order: natamycin and miconazole equally, itraconazole, and ketoconazole, although no significant difference was found among drugs. Fungi were significantly less susceptible to fluconazole (P0.0001) than to the other 4 drugs.Initial antifungal therapy with topically applied natamycin, miconazole, itraconazole, or ketoconazole is recommended for ulcerative keratomycosis in horses in the subtropical environment of Florida.Specific antifungal treatment of horses with ulcerative keratomycosis should be based on history, results of ophthalmic examination, cytologic findings, isolation of the pathogenic fungus, and known prevalence of unique ocular fungi in specific geographic areas. In vitro antifungal susceptibility testing may be most beneficial in aiding documentation of pharmacologic susceptibility patterns of fungi in specific geographic regions.