Mycotoxins are putative virulence factors of fungi that play an important role in the pathogenesis of fungal infections. Mycotoxin production has been used as a diagnostic marker for the early diagnosis of fungal diseases. Using high-performance liquid chromatography, we investigated whether the fungal strains recovered from eye tissue samples obtained from patients with ocular mycoses produced the mycotoxin xanthomegnin. We tested 62 well-characterized strains of fungi, including Aspergillus spp. (n = 14), Exophiala spp. (n = 9), Fusarium spp. (n = 15), and several molds (n = 24). All isolates were identified to the species level using PCR and DNA sequencing of rRNA genes. We detected xanthomegnin activity (0.02 A mu g/ml) in one of the three Aspergillus flavus strains. However, we were unable to detect xanthomegnin in any of the other 61 fungal strains. Our result suggests that xanthomegnin production was infrequent in fungal strains recovered from patients with ocular mycoses.