BACKGROUND Overactive bladder syndrome (OABS) is a common disorder that encompasses urgency, frequency, and nocturia. Various methods of therapies have been utilized worldwide. Despite progresses, it remains a formidable challenge. Laser treatment has emerged to reliably treat the symptoms complex. This study was intended to evaluate the effect of fractional CO2 laser on the main symptoms of OABS. MATERIALS AND METHODS A prospective pre-and post-intervention pilot study was conducted on 31 female subjects recruited from a general hospital in Tehran, Iran. They were treated with 3 laser applications at one-month interval using the intravaginal fractional micro-ablative CO2 laser. The study measures were urgency, frequency, nocturia, leakage, International Consultation on Incontinence Overactive Bladder (ICIQ-OAB), Urogenital Distress Inventory Short Form (UDI-6), and Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire (PISQ-12), which were examined at baseline and every month thereafter up to the end of the laser treatment sessions. RESULTS The findings indicated that statistically significant differences occurred in the ICIQ-OAB score and urgency during treatment sessions as compared to the baseline (p<0.05). Laser therapy led to marked improvements in the UDI-6 score only at month 3 (p=0.001). Nocturia, frequency, leakage, and PISQ-12 score did not ameliorate upon laser administration (p>0.05). CONCLUSION In this pilot study, it was found that fractional CO2 laser could be effective in the amelioration of some OABS symptoms in menopausal women. Further investigations with a larger sample size and long-term follow-ups are required to substantiate these preliminary findings.