This study aimed to assess the effect of laser frenectomy on the closure of midline diastema without orthodontic treatment in mixed and permanent dentitions. The study included 14 patients (6 children in the mixed dentition and 8 adolescents and adults in the permanent dentition) with midline diastema > 1mm. Frenectomy was conducted by a diode laser (810 nm, 3 W, CW). The amount of midline diastema was measured on the study models before, and 1, 3, and 6 months after frenectomy using a digital caliper. The degree of perceived pain was recorded using a Visual Analogue Scale (VAS) on the first and second nights after operation. There was no significant difference in the amount of midline diastema between different time periods (p=0.06), the two age groups (p=0.35), and the two genders (p=0.27). Furthermore, no significant difference was found regarding the pain level between the two groups either on the first (p=0.65) or second (p=0.65) nights. Laser frenectomy leads to slight and insignificant reduction of midline diastema in mixed and permanent dentitions therefore the application of other adjuncts such as orthodontic intervention is required for diastema closure. The use of diode laser is advocated as an appropriate tool with little pain and discomfort after application in patients needing frenectomy.