Various antimicrobials were used in the Neonatal Intensive Care Unit (NICU) based on the patient's health and the results of their bacterial culture. One of the antibacterial options in the NICU is aminoglycoside. Aminoglycosides, especially amikacin and gentamicin, are often used as single or combination therapy in the treatment of neonatal patient infections. The aim of this study was to describe the profile of antimicrobials used as single and/or combination therapy with aminoglycosides in NICU patients in one of hospital in Yogyakarta. This study was conducted using a descriptive method with a cross-sectional design on medical records data in neonatal patients receiving aminoglycoside therapy (amikacin and gentamicin) while undergoing inpatient care at the NICU, one of hospital in Yogyakarta, from 2017 to 2018. The results of this study obtained 171 patients who met the inclusion and exclusion criteria. There were 120 (70.2%) who were 0 days old when admitted to the NICU, 101 (59.1%) of the patients treated had very low birth weight, and 108 patients (63.2%) were born prematurely. Empirical antimicrobial therapy was used by 159 patients, and the rest used empirical and definitive antimicrobial therapy. The most commonly prescribed antimicrobial was a combination of ampicillin and gentamicin antimicrobials in 158 patients (42%). The antimicrobials most commonly used in NICU patients as therapy are ampicillin and gentamicin.