To compare the ocular surface (OS) microbial communities and diversity between dry eye (DE) and non-DE (NDE). Furthermore, we compared meibomian gland dysfunction (MGD) and non-MGD (NMGD) among DE subjects. The V3-V4 region of 16S rRNA gene high-throughput sequencing was performed in the conjunctival swab samples to investigate the composition of the OS bacterial community in DE (n=35) and NDE (n=54) and compared the composition of MGD (n=25) and NMGD (n=10) among DE subjects. Deep sequencing of OS 16S rDNA from DE (n=35) and NDE (n=54) demonstrated great a difference in alpha and beta diversity between the OS bacterial flora (P < 0.05). The similar OS microbial structures were shown at the phylum and genus levels by bioinformatics analysis between them, and in LEfSe (linear discriminant analysis effect size) analysis, Bacteroidia and Bacteroidetes were enriched in DE, while Pseudomonas was plentiful in NDE (linear discriminant analysis [LDA] > 4.0). Among the DE group, there was no significant difference in α and β diversity between MGD and NMGD (P > 0.05). Surprisingly, Bacilli was the dominant microbe in MGD, and Bacteroidetes was the superior bacteria in NMGD among DE subjects (LDA > 4.0). Different diversity of OS bacteria composition between DE and NDE and the altered diversity of OS bacteria may play an important role in DE. Moreover, the lower dominance of OS bacteria in DE may be associated with the occurrence and development of DE. Although there was no significant difference in alpha and beta analysis, the OS dominant microbe between MGD and NMGD among DE was different.