Backgrounds: Neutropenia can be associated with life-threatening infections. Gram negative and staphylococcal infections are the most common pathogens. The spectrum of bacterial isolates has changed considerably over the past four decades. The objective of the present study was to evaluate the pattern of bacterial and fungal infections in neutropenic pediatric patients. Methods: A non-randomized descriptive and cross-sectional study involving 100 hospitalized children was carried out at the emergency and pediatric hematology and oncology units of hospitals affiliated to Mashhad University of Medical Sciences from September 2004 to September 2005. Neutropenic children younger than 12 years old with clinical signs of infection and/or fever were enrolled in the study. Results: The study comprised of 100 febrile and/or infected neutropenic episodes occurring in 57 male and 43 female children younger than 12 years old with a mean age of 4.55±3.33 years. A total of 87 pathogens were cultured: 37 (42.5%) from urinary tract and 50 (57.5%) from other sites; 54 (62.1%) were gram-negative bacteria, 21 (24.1%) were gram-positive bacteria, and 12 (13.8%) were fungus. Pseudomonas aeruginosa and staphylococcus aureus were the most frequent gram-negative and gram-positive isolates respectively. Candida spp. was the only isolated fungus. Acute lymphoblastic leukemia was the most common disease encompassing 33% of all cases. Conclusion: As the patterns of isolates in neutropenic patients are not the same in different parts of the world and gram-negative organisms were still the most common pathogens isolated in our study population, therapeutic adjustments for empirical antibiotic therapy are likely to be focused on gram-negative pathogens.