Objective To characterize vaginal microecology and target reproductive tract pathogen DNA detection in patients with adenomyosis (AM) and to identify associated factors. Methods This case-control study included 50 women with AM treated at The Second Affiliated Hospital of Henan University of Science and Technology from November 2024 to November 2025 and 48 women without AM examined during the same period. Morphological indicators of vaginal microbiota, functional indicators[vaginal pH value, H2O2, leukocyte esterase (LE) and sialidase (SNA)], Nugent scores, and target pathogen DNA positivity were compared between the two groups. Variables were selected according to the number of outcome events, clinical relevance, and intervariable correlations for multivariable binary Logistic regression, and the discriminatory performance of the model was evaluated. Results Histories of infertility and intrauterine procedures were more frequent in the AM group than in the control group (all P＜0.05), whereas the other baseline characteristics were comparable (all P＞0.05). The AM group showed higher proportions of abnormal flora density, flora diversity, dominant flora, Nugent score, vaginal pH, and H2O2, as well as higher LE and SNA positivity (all P＜0.05). DNA positivity for Ureaplasma parvum serovars 1, 3, and 6 and Ureaplasma urealyticum were also higher in the AM group (all P＜0.05). Multivariable Logistic regression identified a history of intrauterine procedures, abnormal dominant flora, abnormal vaginal pH, and abnormal H2O2 as factors independently associated with AM (all P＜0.05). The combined model yielded an area under the receiver operating characteristic curve of 0.821 (95%CI: 0.734-0.900), with 78.00% sensitivity and 77.08% specificity. Conclusion Vaginal microecological abnormalities and DNA positivity for selected reproductive tract pathogens are more frequently observed in patients with AM. A history of intrauterine procedures, abnormal dominant flora, abnormal vaginal pH, and abnormal H2O2 are independently associated with AM; however, the above results do not yet establish a causal relationship and require further prospective studies for verification.