Background Female urinary microbiota are associated with urgency urinary incontinence and response to medication. The urinary microbiota of women with stress urinary incontinence has not been described. Objective We sought to study the cross-sectional relationships between urinary microbiota features and demographic and clinical characteristics of women undergoing stress urinary incontinence surgery. Study Design Preoperative urine specimens were collected from women without urinary tract infection and were available from 197 women (174 voided, 23 catheterized) enrolled in a multicenter prospective randomized trial, the Value of Urodynamic Evaluation study. Demographic and clinical variables were obtained including stress and urgency urinary incontinence symptoms, menopausal status, and hormone use. The bacterial composition of the urine was qualitatively assessed by sequencing the bacterial 16S ribosomal RNA gene. Phylogenetic relatedness and microbial alpha diversity were compared to demographics and symptoms using generalized estimating equation models. Results The majority of 197 urine samples (86%) had detectable bacterial DNA. Bacterial diversity was significantly associated with higher body mass index (P =.02); increased Medical, Epidemiologic, and Social Aspects of Aging urge index score (P =.04); and hormonal status (P <.001). No associations were detected with stress urinary incontinence symptoms. Increased diversity was also associated with a concomitant lower frequency of Lactobacillus in hormone-negative women. Conclusion Women undergoing stress urinary incontinence surgery have detectable urinary microbiota. This cross-sectional analysis revealed that increased diversity of the microbiota was associated with urgency urinary incontinence symptoms, hormonal status, and body mass index. In contrast, the female urinary microbiota were not associated with stress urinary incontinence symptoms.