Eye drops used for diagnostic mydriasis may produce systemic side effects in preterm infants. Studies on the pupil dilating and systemic effect of various mydriatic agents yielded conflicting results. We conducted a prospective randomized double-blind study on the systemic effect of two mydriatic eye-drop combinations. Thirty-nine preterm infants were randomly assigned to two groups. An eye-drop combination of 2.5% phenylephrine and 0.5% tropicamide (group D) was compared with the combination of 0.5% cyclopentolate and 0.5% tropicamide (group F). Either eye-drop combination was followed by 0.5% tropicamide given 20 minutes later. Heart rate (HR) and the systolic, mean, and diastolic blood pressure (BP) were recorded before and after eye-drop instillation and after ophthalmoscopy. A control session with NaCl eye drops was added for each infant. A significant increase of BP and HR peak values was observed within 7 to 10 minutes after the cyclopentolate/tropicamide combination only. On the other hand, the mydriatic effect of the phenylephrine/tropicamide combination was significantly superior to that of the cyclopentolate/tropicamide combination. We recommend the combination of 2.5% phenylephrine and 0.5% tropicamide to achieve a sufficient diagnostic mydriasis without systemic side effects in preterm infants.