To evaluate the safety and efficacy of transvaginal fractional microablative CO2 laser therapy (MLT) on urinary symptoms in postmenopausal women with genitourinary syndrome of menopause (GSM) in relation to the timing of their onset; if prior to or after menopause. Secondary, the efficacy of MLT on vulvovaginal atrophy (VVA)-related symptoms. This is a retrospective analysis of prospectively collected data. Postmenopausal women affected by at least one urinary symptom (urinary frequency, urgency incontinence, stress urinary incontinence) and VVA symptom each (dryness, dyspareunia, itching, burning) were enrolled. Our population was divided into two groups in relation to the onset of urinary symptoms, prior to or after menopause. Women were treated with three CO2 MLT laser sessions, administered at a 4-week interval. For urinary symptoms evaluation, we used the following disease-specific questionnaires previously validated in Italy: the Urogenital Distress Inventory score (UDI-6) and the International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF). For each single VVA symptom, we assessed patient's severity perception with a 10-point visual analog scale. Time points of the study were at entry (T0) and at 16 weeks since the first treatment (T1). Collected data were analyzed with the Pearson χ 2 test for categorical variables and the Wilcoxon rank-sum test (for non-normally distributed data), and statistical significance was defined with a P-value <0.05. Sixty-one women were enrolled in this study. Overall, at T1 MLT brought to a significant improvement in urinary symptoms (P < 0.05) in women with urinary symptoms started after the menopause (Group B), contrary to the ones with urinary symptoms started before the menopause (Group A). Specifically, urinary frequency significantly improved only in Group B (P < 0.05), while urgency incontinence, significantly reduced in both groups (P < 0.05). Stress urinary incontinence did not significantly improve in both groups (P > 0.05). Secondary, all VVA symptoms showed a statistically significant improvement (P < 0.05) at 16 weeks from baseline; no differences were registered between groups. No adverse events were recorded. This study confirms the safety and efficacy of CO2 MLT for GSM symptoms. When urinary symptoms are considered, it seems that MLT might have a higher efficacy when symptoms started after menopause, in particular when they are part of the OAB syndrome. Lasers Surg. Med. © 2021 Wiley Periodicals LLC.