Most patients who undergo hematopoietic stem cell transplantation develop neutropenic fever and are at high risk for developing potentially life-threatening infections. β-lactam antibiotics remain the cornerstone for initial empiric treatment of neutropenic fever. In cancer patients with allergy or intolerance to β-lactams, guidelines recommend using aztreonam with vancomycin (AV) for neutropenic fever treatment. To date, the efficacy of AV for the treatment of neutropenic fever during stem cell transplantation is unknown. A retrospective study was conducted to identify hematopoietic stem cell transplantation recipients who were initially treated with concomitant AV for neutropenic fever between 2007 and 2013. Febrile neutropenia was classified as neutropenia with unexplained fever, neutropenic fever with a local source of infection, or neutropenic fever with a microbiologically documented infection. Seventy-six patients were identified who received AV as initial treatment for neutropenic fever over the study period. Responses to AV for neutropenia with unexplained fever (n = 41), febrile neutropenia with local site of infection (n = 11 [pneumonia = 9, other = 2]), and neutropenic fever with microbiologically documented infection (n = 34) were 75%, 55% (45% pneumonia), and 46% respectively. Infection-related mortality was 5%. Aztreonam with vancomycin was effective in treating neutropenia with unexplained fever. For patients with neutropenic fever and local source or microbiologically documented infection, alternative antibiotic treatments should be considered.