We evaluated the ototoxic effect of aminoglycosides on the outer hair cells of newborns in a neonatal intensive care unit (NICU) by means of distortion-product otoacoustic emissions (DPOAE) testing. Our study population was made up of 164 newborns who were divided into three groups: group A consisted of 105 infants who were given aminoglycoside therapy (either gentamicin or amikacin, or a combination of the two) as treatment for suspected or proven bacterial infection and septic states; group B included 30 newborns who were not given an antibiotic or who were given an antibiotic other than an aminoglycoside; group C, a control group, was made up of 29 healthy neonates who were hospitalized in the well-baby nursery. All the neonates underwent DPOAE testing in both ears (the f2 primary tone was presented at 2.0, 2.5, 3.2, and 4.0 kHz). We found that 41 patients in group A (39.0%) and 13 in group B (43.3%) failed the DPOAE test in one or both ears; the difference between these two groups was not statistically significant (p = 0.673). In group C, the DPOAE fail rate was 13.8% (4 newborns). In group A, there was no statistically significant association between the pass/fail rate and the specific aminoglycoside that was administered, or in the duration of antibiotic treatment, the number of doses, and the size of the mean daily dose and the mean total dose. In clinical practice, DPOAE testing is a sensitive method of evaluating the integrity of the outer hair cells in the basal turn of the cochlea after exposure to ototoxic drugs such as aminoglycosides. However, our study did not demonstrate that the aminoglycosides had any ototoxic effect on the hearing of neonates in the NICU.