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Histopathological examination of the nail matrix is essential for diagnosing longitudinal melanonychia (LM) and distinguishing benign conditions from melanoma. However, traditional biopsy techniques can be invasive, technically complex, and risk permanent nail dystrophy. This study evaluates the feasibility, diagnostic accuracy, and cosmetic outcomes of a minimally invasive CO₂ laser-assisted biopsy technique. We conducted a retrospective case series on patients with single-digit LM who underwent CO₂ laser-assisted nail matrix biopsy at our dermatology department between January 2023 and December 2023. A small square-shaped window was created in the nail plate using a CO₂ laser, avoiding proximal nail fold incisions. A 3-5 mm punch biopsy was performed on the distal nail matrix, and the excised nail plate was repositioned as a protective biological dressing. Patients were followed up for healing, complications, and cosmetic outcomes. Ten patients (six females, four males, median age: 39 years) were included. Biopsy samples were sufficient for diagnosis in all cases, identifying melanocytic activation (50%) and matrix nevi (40%), with one inconclusive case. Three patients required reintervention due to previous insufficient biopsy samples or recurrence. Follow-up ranged from 6 to 15 months, with mild postoperative nail changes in 50% of cases, more common in fingernails and reinterventions. No severe nail dystrophy occurred. CO₂ laser-assisted biopsy is a minimally invasive, effective technique for LM diagnosis, preserving nail structure while reducing complications. Larger studies are needed to confirm its long-term benefits over conventional methods.