Background Anti-human T-lymphocyte immunoglobulin is commonly used as prophylaxis for graft-versus-host disease after allogeneic hematopoietic stem cell transplantation from unrelated donors. The studies according to optimum dose of ATLG especially in pediatric patients are limited. Patients and Methods Outcomes of 99 pediatric patients diagnosed with nonmalignant diseases, who received ATLG as GVHD prophylaxis for matched unrelated donor HSCT at a dose of 10 mg/kg (group 1), 20 mg/kg (group 2), and 30 mg/kg (group 3), were analyzed retrospectively. Results The incidences of acute and chronic GVHD were statistically not different between three groups (p = .20 and p = .13), but we did not observe chronic GVHD in group 3 patients. Cox regression analysis showed that ATLG dose of 10 mg/kg (p = .007) and severe acute GVHD (p = .001) were significant prognostic factors for inferior overall survival. Although ATLG dose of 10 mg/kg is effective in pediatric patients on acute and chronic GVHD prevention, TRM and overall survival were superior in ATLG doses >= 20 mg/kg (p = .04 and p = .037) with no difference between 20 and 30 mg/kg. Conclusion Although ATLG dose of 10 mg/kg is effective in pediatric patients on acute and chronic GVHD prevention and safe from the point of infection, TRM and OS were superior in ATLG doses >= 20 mg/kg with no difference between 20 and 30 mg/kg. These observations should be supported with other multicenter prospective studies including larger patient population.