Objective To analyze the characteristics of intestinal flora in children with chalazion, and explore the relationship between the composition of intestinal flora and chalazion.Methods Fecal samples were collected from 21 children with chalazion and 26 healthy children. DNA was extracted from the fecal samples, and the 16S rRNA sequences were detected by the second generation sequencing technology. The results were used to compare the composition of the microbiome between children with chalazion and healthy controls.Results There was no significant difference in intestinal flora diversity or relative abundance between or in the 2 groups according to the alpha and beta diversity. Eleven differential species were screened out by the linear discriminant analysis effect size (LEfSe) analysis. ANOSIM analysis, t test and metastats analysis were used to compare the intestinal flora of the healthy control group and chalazion group. The results of t test showed 7 differential species, and metastats analysis results showed 40 differential species. Gut_metagenome and human_gut_metagenome were common differential species at the species level. Finally, corrplot analysis showed that gut_metagenome was strongly correlated with the number, ulceration and postoperative recurrence of chalazion in children (r=0.70, 0.76, 0.85, all P < 0.01).Conclusion There is no significant difference in intestinal diversity or relative abundance between children with chalazion and healthy children, but there are significant differences in some bacterial species. Gut_metagenome may be a microbiological indicator which is independent of clinicopathologic factors, but is associated with chalazion disease.