Purpose: To evaluate the effects of benzalkonium chloride (BAK) on the blood-aqueous (BAB) and blood-retinal barriers (BRB) of pseudophakic eyes. Methods: Prospective, randomized, investigator-masked, comparative study. Patients were randomly assigned to preservative-free artificial tears or BAK-preserved artificial tears. One drop of artificial tears was instilled 4 times a day in the study eye, starting the day after randomization for 30 days. Anterior chamber flare was assessed by a laser flare meter (LFM) and macular thickness measurements were obtained with optical coherence tomography, before, 15, and 30 days after randomization. Results: A total of 44 healthy eyes of 44 pseudophakic volunteers were recruited. There were no significant differences regarding demographics (age, gender, and race distributions) and clinical characteristics (eye, mean intraocular pressure, and mean best-corrected visual acuity) between the 2 groups (P > 0.05). No significant differences in baseline mean LFM values were observed (P = 0.262). However, we detected a statistically significant increase in mean LFM measurements in the BAK-preserved group (11.4 +/- 5.1 ph/ms) (P = 0.017) after 15 days. After 30 days, the BAK-preserved group maintained significantly higher flare values (11.9 +/- 5.9 ph/ms) compared with baseline (P = 0.043). On the other hand, the preservative-free group showed mean flare values of 8.4 +/- 2.5 ph/ms, not significantly different from those obtained at baseline (P = 1.00). We observed no statistically significant change in macular thickness measurements at days 15 and 30 in either group (P > 0.05). Cystoid macular edema was not detected in this series. Conclusions: Our results suggest that a short-term exposure to BAK can cause disruption of the BAB, without altering the BRB in pseudophakic eyes.