Purpose To evaluate the effect of topical tropicamide 1% and phenylephrine 2.5% instillation on macular and peripapillary microvasculature measurements with optical coherence tomography angiography (OCTA). Methods Forty eyes of 40 consecutive healthy adults with no known systemic or ocular disease were recruited for this prospective consecutive case study. After complete ophthalmological examination, all patients underwent OCTA measurements (OptoVue Inc, Freemont, CA, USA) to assess foveal avascular zone (FAZ) area, FAZ perimeter, acircularity index of FAZ, foveal density, vessel density of superficial and deep capillary plexus and peripapillary capillary plexus. 6 x 6 mm macular and 4.5 x 4.5 mm peripapillary OCTA images were undertaken before and 30 min after instillation of tropicamide (20 eyes) or phenylephrine (20 eyes) instillation to the right eye, and these were compared to each other and to fellow control eye. Results 15 male and 25 female patients with a mean age of 43.3 (18-60) years were recruited for the study. Superficial, deep and peripapillary capillary plexus measurements of tropicamide 1% and phenylephrine 2.5% instilled right eyes and left control eyes were similar before and 30 min after instillation (P > 0.05 for all). FAZ assessment tool variables were also similar before and after instillation (P > 0.05 for all) for both eyes. Conclusion Topical pupillary dilatation with tropicamide 1% and phenylephrine 2.5% did not affect macular and peripapillary OCTA measurements. Follow-up OCTA images in retina and glaucoma patients can be captured with a dilated or undilated pupil which seems not to be affected by tropicamide or phenylephrine.