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AIM: To investigate the ocular surface microbiome profile of patients with fungal keratitis (FK) through bacterial 16S rDNA sequencing. METHODS: The swab samples were collected from 8 patients with FK (Group 1 from the corneal ulcer, Group 2 from the conjunctival sac of the infected eyes, and Group 3 from the conjunctival sac of the fellow eyes) and 10 healthy eyes (Group 4 from the conjunctival sac). Bacterial 16S rDNA V4-V5 region sequencing was performed to characterize the bacterial communities on the ocular surfaces of the patients with FK. RESULTS: Our metagenomic data showed that 97% of the sequence reads were categorized into 245 distinct bacterial genera, with 67.75±7.79 genera detected in Group 1, 73.80±13.44 in Group 2, 74.57±14.14 in Group 3, and 89.60±27.49 in Group 4. Compared with the healthy eyes (Group 4), both infected (Groups 1 and 2) and fellow eyes (Group 3) of the patients with FK showed reduced bacterial diversity and altered ocular surface microbiota compositions, with lower abundance of Corynebacterium and Staphylococcus and higher abundances of Pseudomonas, Achromobacter, Caulobacter and Psychrobacter. CONCLUSION: Our report depicts the altered ocular surface bacterial community structures both in the affected and fellow eyes of patients with FK. These changes may contribute to the pathogenesis of FK or the increased risk for FK.