Introduction: Demodex and bacteria are both components of the ocular surface micro-ecology, constituting a complex interaction. This study aims to explore how ocular surface Demodex infestation (DI) affects ocular surface microbial communities and diversity. Methods: We recruited 255 subjects, and examined the correlation between ocular surface mite infestation and clinical indicators such as age, blood glucose level, dry eye symptoms, and blood pressure. 16S rRNA sequencing was performed on the conjunctival swab samples of 14 patients with ocular DI (P group) and 17 healthy people (N group). For further analysis, the subjects were divided into four subgroups, i.e. N-NMGD (n = 11), N-MGD (n = 6), P-NMGD (n = 6), and P-MGD (n = 8), according to meibomian gland dysfunction (MGD) or no MGD (NMGD). Results: There was no difference in the a-diversity of ocular surface microbial communities between the DI and healthy control groups. In linear discriminant analysis effect size (LEfSe), there were more Acinetobacter, Novosphingobium, and Anoxybacillus in the DI group and fewer Novosphingobium, Lactobacillus, and Candidatus Microthrix in the healthy control group. P-NMGD had more Thermaceae and fewer Pseudomonas than P-MGD. There were more Bacteroidetes in N-NMGD than in N-MGD. The Xiaotian Liang and Yingli Li contributed equally to the manuscript.