pneumonia is a life-threatening opportunistic infection in children receiving immunosuppressive chemotherapy. Without prophylaxis, up to 25% of pediatric oncology patients receiving chemotherapy will develop pneumonia. Trimethoprim-sulfamethoxazole is the preferred agent for prophylaxis against pneumonia. Pentamidine may be an acceptable alternative for pediatric patients unable to tolerate trimethoprim-sulfamethoxazole. A retrospective review was conducted of pediatric oncology patients who received ≥1 dose of pentamidine for pneumonia prophylaxis between January 2007 and August 2014. Electronic medical records were reviewed to determine the incidence of breakthrough pneumonia or discontinuation of pentamidine associated with adverse events. A total of 754 patients received pentamidine prophylaxis during the period. There were no cases of probable or proven pneumonia, and 4 cases (0.5%) of possible pneumonia. The incidence of possible breakthrough pneumonia was not significantly different between subgroups based on age (<12 months [1.7%] versus ≥12 months [0.4%], = 0.3), route of administration (aerosolized [0%] versus intravenous [1.0%], = 0.2), or hematopoietic stem cell transplant status (transplant [0.4%] versus no transplant [0.8%], = 0.6). Pentamidine was discontinued due to an adverse drug event in 23 children (3.1%), more frequently for aerosolized than for intravenous administration (7.6% versus 2.2%, respectively, = 0.004). Intravenous or inhaled pentamidine may be a safe and effective second-line alternative for prophylaxis against pneumonia in children with cancer receiving immunosuppressive chemotherapy or hematopoietic stem cell transplantation.