Purpose: To investigate the influence of topical cyclopentolate 1%, as an anti-muscarinic mydriatic agent, on the peripapillary and macular microvasculature by optical coherence tomography angiography (OCT-A) in healthy adults. Methods: A total of 41 healthy adults without any systemic or ocular disease were enrolled for this prospective consecutive study. All patients underwent OCT-A measurements (OptoVue Inc., Freemont, CA, USA) to assess optic disc status for radial peripapillary capillary network (whole image, inside disc, and peripapillary capillary densities), and superficial and deep capillary plexus whole, foveal, parafoveal and perifoveal densities, and foveal avascular zone (FAZ) densities. Foveal retinal thicknesses and all quadrant retinal fiber layer thicknesses were also assessed. The 4.5 mm × 4.5 mm peripapillary and 6 mm × 6 mm macular OCT-A images were undertaken before and 30 min after instillation of topical cyclopentolate 1% to the right eyes. Results: The mean age of subjects was 38.14 ± 14.10 years. All macular, optic disc, and FAZ densities, foveal retinal thicknesses, average, and all quadrant retinal fiber layer thicknesses were statistically similar between baseline and after administration of topical cyclopentolate 1% ( P > 0.05). Conclusion: The current study demonstrated that pupillary dilation with topical cyclopentolate 1% seems to have no statistical effect on macular and peripapillary OCT-A measurements of healthy adults.