Introduction: It has been hypothesized that the urinary microbiome might play an important role in OAB. Studies have been conducted on the association between OAB symptoms and the microbiome, although a possible causality still has to be determined. Material and Methods: In this study, 12 female patients, ≥18 years of age, with ‘OAB DO+’ and 9 female patients with ‘OAB DO-’ were included. Patients were excluded if they met one of the following exclusion criteria: bladder tumors and previous bladder operations; sacral neuromodulation; injection of Botox in the bladder; and TOT or TVT operations. Urine samples were collected and stored with patient informed consent and with the approval of the Hospital Ethical Review Board (Arnhem–Nijmegen). All OAB patients underwent urodynamics before collecting urine samples, and the diagnosis of detrusor overactivity was confirmed by two individual urologists. In addition, samples from 12 healthy controls who did not undergo urodynamic evaluation were analyzed. The 16S rRNA V1–V2 region amplification and gel electrophoresis were used to determine the microbiota. Results: 12 of the OAB patients had DO shown on their urodynamic studies; the remaining 9 patients had a normoactive detrusor on their urodynamic measurements. Overall, there were no substantial differences among the demographic characteristics of the subjects. The samples were classified as the following: 180 phyla, 180 classes, 179 orders, 178 families, 175 genera, and 138 species. The least commonly observed phyla were Proteobacteria, with an average presence of 10%, followed by Bacteroidetes with 15%, Actinobacteria with 16%, and Firmicutes with 41%. Most of the sequences could be classified according to the genus level for each sample. Discussion: Significant differences were observed in the urinary microbiome of patients with overactive bladder syndrome who have detrusor overactivity on urodynamics compared to OAB patients without detrusor overactivity and matched controls. OAB patients with detrusor overactivity have a significantly less diverse microbiome and show a higher proportion ofLactobacillus, particularlyLactobacillus iners. The results imply that the urinary microbiome could be involved in the pathogenesis of a specific phenotype of OAB. The urinary microbiome could be a new starting point to study the causes and treatments of OAB.