Objective To investigate the relationship between the bladder microbiota and overactive bladder (OAB) symptom severity. Design Cross-sectional study of female OAB patients who contributed catheterised urine samples, completed validated symptom questionnaires, and provided demographic data. Setting Nanfang Hospital, Southern Medical University. Population Adult women with overactive bladder symptom. Methods 70 OAB patients were recruited, finished symptoms questionnaires, demographic data, and contributed catheterised urine samples that were analysed by 16S rRNA gene sequencing. Main outcome measures Associations between the total/sub scores of Overactive Bladder Symptom Score (OABSS) and bladder microbiota (diversity, overall community structure, and specific organisms). Results We found that bacterial diversity (Simpson index, P = 0.024) and richness (Chao1, P = 0.023) of mild patients (OABSS ≤5, N = 17) were lower than those of moderate/severe patients (OABSS > 5, N = 53). And bacterial communities of two groups were significantly different. Further, there were positive correlations between scores of OABSS and both richness (Chao1, P = 0.002) and diversity (Shannon index, P = 0.044) of urinary microbiome. Some bacterial genera (e.g., Porphyromona and Prevotella) were significantly related to sub-symptoms of OAB. Conclusion This cross-sectional analysis revealed that increased diversity and richness of the bladder microbiota was associated with worse OAB severity, and specific urinary dysbiosis may constitute an etiological factor in exacerbating functional bladder disorders. Keywords overactive bladder; LUTS; urinary microbiome; urinary microbiota; bladder microbiota Funding National Natural Science Foundation of China (Grant No. 81870522) Tweetable abstract Bladder microbiota is associated with female OAB severity.