Dysfunctional voiding (DV) in women is a common disorder that causes bladder outlet obstruction (BOO) and may aggravate overactive bladder (OAB) symptoms. The diagnosis of DV relies on clinical alertness and videourodynamic study (VUDS). In this pilot study, we aimed to explore the diagnostic potential of urinary biomarkers in women with DV. Women who visited our institute for the management of OAB were enrolled in this prospective study. Urine samples were collected before treatment and urinary proteins were quantified using enzyme-linked immunosorbent assay (ELISA) kits. Patients with DV and patients with normal tracings were included in the study and control group, respectively. The patients with DV were further divided into two subgroups: those with and those without detrusor overactivity (DO). VUDS parameters and urinary protein levels were compared between each subgroup. A total of 67 women were enrolled. There were 47 patients with VUDS-diagnosed DV and 20 patients in the control group. Urinary 8-OHdG level (32.65 vs. 15.59, p < 0.001) and urinary TNF-α level (1.43 vs. 0.83, p = 0.031) were significantly higher in the DV group, and urinary IL-2 level (0.29 vs. 0.83, p < 0.001) was significantly lower in the DV group. Among the DV patients, urinary PGE2 level was higher in the DO group (240.4 vs 149.6, p = 0.049). In women with DV, urinary 8-OHdG level is elevated compared to women with normal VUDS. Urinary PGE2 level is elevated in patients with DV and DO. These finding suggests increased reactive-oxidative stress response and detrusor hyperactivity in DV patients. Urinary biomarkers can serve as potential non-invasive diagnostic tools in women with OAB.