Neutropenia that occurs in hematologic malignancies or solid tumors may increase the risk of infection and lead to morbidity and mortality. This study aims to investigate the microbiological profile and antibiotic susceptibility pattern in children with malignancy and febrile neutropenia (FN). A cross-sectional study was conducted from January 2017 to June 2021 using medical records from the pediatric inpatient hematology-oncology ward of Dr. Soetomo General Hospital. The inclusion criteria are children with malignancy aged ≤ 18 years, experiencing a febrile episode of neutropenia, and were examined for blood culture at the onset of FN. A total of 291 children were eligible patients. Out of 291, 65 of them had a bacterial infection from blood cultures. Out of 65, almost 57% were dominated by Gram-negative bacteria (GNB). Gram-positive bacteria (GPB) that are often recognized are Staphylococcus hominis, Staphylococcus epidermidis, and Staphylococcus aureus. Meanwhile, the GNB are Pseudomonas aeruginosa and Klebsiella pneumoniae ESBL+. The majority of GNB is almost 100% resistant to ampicillin meanwhile, GPB also reported resistance to β-lactamase. We found no correlation between microorganism infection and malignancy. GNB and GPB are partially resistant to commonly used antibiotics, especially β-lactamases and ampicillin. Alternative therapies are needed to resolve this matter.