The ocular surface possesses its own bacterial microbiota. Once given a chance, opportunistic pathogens within ocular microbiota may lead to corneal infection like bacterial keratitis (BK). To reveal the possible factor that makes people vulnerable to BK from the perspective of ocular bacterial microbiota, as well as to compare diagnostic information provided by high-throughput 16S rDNA sequencing and bacterial culture, 20 patients with BK and 42 healthy volunteers were included. Conjunctival swabs and corneal scrapings collected from the diseased eyes of BK patients were subjected for both high-throughput 16S rDNA sequencing and bacterial culture. Conjunctival swabs collected from the normal eyes of BK patients and healthy volunteers were sent only for sequencing. For identifying the pathogens causing BK, high-throughput 16S rDNA sequencing presented a higher positive rate than bacterial culture (98.04% vs. 17.50%), with 92.11% reaching the genus level (including 10.53% down to the species level). However, none of the sequencing results was consistent with the cultural results. The sequencing technique appears to challenge culture, and could be a complement for pathogen identification. Moreover, compared to the eyes of healthy subjects, the ocular microbiota of three sample groups from BK patients contained significantly less Actinobacteria and Corynebacteria (determinate beneficial symbiotic bacteria), but significantly more Gammaproteobacteria, Pseudomonas, Bacteroides, and Escherichia-Shigella (common ocular pathogenic bacteria). Therefore, it is speculated that the imbalance of protective and aggressive bacteria in the ocular microbiota of healthy people may trigger susceptibility to BK. Based on this speculation, it seems promising to prevent and treat infectious oculopathy through regulating ocular microbiota.