Opportunistic mycoses have emerged as important causes for morbidity and mortality in pediatric cancer patients, particularly in those with intensively treated hematological malignancies, allogeneic hematopoetic stem cell transplantation, and aplastic anemia. The incidence of invasive fungal infections in these settings may range from 10 to 25 % despite empirical antifungal therapy with an overall case fatality rate of up to 50 and 75 % depending on the organism. Preventive interventions are thus warranted, including but not limited to chemoprophylaxis with antifungal agents. Effective chemoprophylaxis of invasive Candida infections with a long-term benefit for overall survival has been demonstrated in patients with allogeneic bone marrow transplantation. However, its benefit in other high-risk populations is less well established, and a clearly effective approach to chemoprophylaxis for invasive Aspergillus infections has not been documented in appropriately designed clinical trials. This article reviews epidemiology and current approaches to chemoprophylaxis of opportunistic invasive fungal infections in children and adolescents with cancer and/or stem cell transplantation, and provides evidence-based guidelines for indications and modalities of antifungal prophylaxis and antifungal infection control measures in this population.