Background: Arsenical keratosis, a chronic skin disorder resulting from prolonged exposure to inorganic arsenic, poses a significant public health challenge in arsenic-endemic regions like Bangladesh. Characterized by hyperkeratotic lesions, particularly on the palms and soles, the condition is a known precursor to skin cancer. Current treatment options offer limited efficacy, necessitating the exploration of alternative therapies. Objective: This study aimed to investigate the dose-dependent cytotoxicity of Nigella sativa seed extract and assess its therapeutic potential in the management of palmar arsenical keratosis. Methods: Conducted over 17 months, this observational study involved 32 patients diagnosed with moderate to severe palmar arsenical keratosis from two high-risk unions in Chuadanga, Bangladesh. Nigella sativa seed extract ointment was formulated and applied topically. The primary outcome was the reduction in the size of keratotic nodules, measured before and after 12 weeks of treatment using vernier slide calipers. Arsenic levels in patients’ water and nail samples were analyzed using a modified silver diethyldithiocarbamate method. Statistical analyses were performed using paired t-tests and Pearson correlation. Results: Topical application of Nigella sativa seed extract resulted in a statistically significant 72.8% reduction in mean lesion size (from 55.4 ± 29.8 mm² to 15.1 ± 10.8 mm²; p < 0.0001). No significant correlation was found between lesion size reduction and duration of arsenic exposure (r = -0.28), duration of lesion appearance (r = -0.17), or arsenic concentration in drinking water (r = -0.19). Patient adherence to treatment was high (mean adherence: 94.0 ± 6.6%), and only one case of mild, transient local irritation was reported. Conclusion: The findings demonstrate that Nigella sativa seed extract exhibits dose-dependent cytotoxicity against hyperproliferative keratinocytes, making it a promising, well-tolerated topical therapeutic for palmar arsenical keratosis. Compared to previous treatments, this natural formulation offers superior efficacy and minimal side effects, warranting further investigation through larger clinical trials.