Conjunctival sac microbiome alterations have been reported to be closely associated with many ocular diseases. However, the characteristic of conjunctival sac microbiome in allergic conjunctivitis (AC) was scarcely described. In this study, we aimed to identify the differences of the conjunctival sac microbiome composition in AC patients compared with normal controls (NCs) using high-throughput 16S rDNA sequencing metagenomic analysis. The conjunctival sac microbiome samples from 28 AC patients and 39 NC patients were collected. The V3-V4 region of 16S rRNA gene high-throughput sequencing was performed on the illumina MiSeq platform. Alpha diversity, beta diversity and the relative abundance at the phylum and genus levels were analyzed using QIIME. Alpha diversity demonstrated by Chao1, Observed_species and PD_whole_tree indexes did not show significant difference between the AC and NC groups, while the Shannon index was higher in the AC group. Beta diversity showed divergent microbiome composition in different groups ( p < 0.005). The top five abundant phyla were Firmicutes, Proteobacteria, Actinobacteriota, Bacteroidota and Cyanobacteria in both groups. The top five abundant genera were Bacillus, Staphylococcus , Corynebacterium, Acinetobacter and Ralstonia in the AC group and Acinetobacter , Staphylococcus , Bacillus , Clostridium_sensu_stricto_1 , Corynebacterium and Geobacillus in the NC group. The Firmicutes/Bacteroidetes (F/B) ratio at the phylum level was similar between groups ( p = 0.144). The Bacillus/Acinetobacter ( B/A ) ratio at the genus level was higher in the AC group ( p = 0.021). The dysbiosis detected in this study might provide further evidence to investigate the mechanism and treatment methods for allergic conjunctivitis.