Objective: To investigate the relationship between high-risk human papillomavirus (HR-HPV) infection and vaginal microecology. Methods: A total of 296 HR-HPV-positive subjects and 316 HR-HPV-negative subjects were selected. The vaginal flora, vaginal microecological indicators, microecological disorders and bacterial vaginosis were compared between the two groups. Results: Compared with the HR-HPV-negative group, the HR-HPV-positive group showed a significant decrease in the bacterial density (II–III) and microbial diversity (II–III) (χ² were 18.548 and 16.693 respectively; all P<0.01). There was no statistical difference in the proportion of dominant bacteria between the two groups (χ²=0.217, P>0.05). Compared with the HR-HPV negative group, the positive rates of hydrogen peroxide, leukocyte esterase and sialidase were significantly increased in the HR-HPV positive group (χ² were 10.560, 8.030 and 12.969 respectively; all P<0.01). There was no statistically significant difference in pH value between the two groups (χ²=0.079, P>0.05). Compared with the HR-HPV-negative group, the ratios of microecological disorders and bacterial vaginosis were significantly increased in the HR-HPV-positive group (χ² were 14.882 and 9.141 respectively; all P<0.01). Conclusion: HR-HPV infection is closely related to vaginal microecological disorders and bacterial vaginosis, maintaining vaginal microecological balance is an effective way to reduce HR-HPV infection and prevent cervical lesions.