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Several studies have shown interstitial cystitis/bladder pain syndrome (IC/BPS), a chronic condition that poses challenges in both diagnosis and treatment, is associated with painful pelvic floor muscles (PFM) and altered neural drive to these muscles. However, its pathophysiology could also involve other alterations in the electrical activity of PFM motor units (MUs). Studying these alterations could provide novel insights into IC/BPS and help its clinical management. This study aimed to characterize PFM activity at the MU level in women with IC/BPS and pelvic floor myalgia using high-density surface electromyography (HD-sEMG). Signals were recorded from 15 patients and 15 healthy controls and decomposed into MU action potential (MUAP) spike trains. MUAP amplitude, firing rate, and magnitude-squared coherence between spike trains were compared across groups. Results showed that MUAPs had significantly lower amplitudes during contractions on the patients’ left PFM, and delta-band coherence was significantly higher at rest on their right PFM compared to controls. These findings suggest altered PFM tissue and neuromuscular control in women with IC/BPS and pelvic floor myalgia. Our results demonstrate that HD-sEMG can provide novel insights into IC/BPS-related PFM dysfunction and biomarkers that help identify subgroups of IC/BPS patients, which may aid their diagnosis and treatment.