Background To determine effects and side effects of topical application of phenylephrine 2.5% and tropicamide 0.5% combination in preterm infants. Methods In this prospective observational study, 60 infants undergoing retinopathy of prematurity (ROP) screening were prospectively observed. Pupillary diameter, blood pressure, heart rate, and oxygen saturation were monitored before and after up to 24 h during ROP screening examinations. Results The mean pupillary diameter 1 h after the instillation of drops was 5.58 +/- 0.75 mm for both eyes. The mean systolic and diastolic pressure and oxygen saturation of infants did not change statistically until the end of the study. The average heart rate decreased by a mean of 4.96 beats/minute from the baseline following eye drops instillation. General condition deterioration, fall in oxygen saturation and bradycardia were observed in 4 infants that already had respiratory distress syndrome. Conclusion The phenylephrine 2.5% plus tropicamide 0.5% drop is effective and safe as mydriatic combination for retinopathy of prematurity screening. In infants with an additional systemic disease such as respiratory distress syndrome, the side effects of mydriatic drops may be more common. Such babies should be kept under close observation.