To evaluate safety and tolerability on the ocular surface of an anti-septic formulation containing 0.6% povidone-iodine (0.6% PVI) for a 4 week period. An observational, prospective study included 20 mild-moderate dry eye disease (DED) patients who enrolled at the Ocular Surface Disease Unit of the University of Messina, receiving 0.6% PVI eye drops for 28 days, 2 drops twice daily (BID). The assessment included the Ocular Surface Disease Index questionnaire; symptoms score (0 = absent to 3 = severe) for burning, ocular dryness, foreign body sensation, watery eyes, tearing, photophobia, and ocular pain; fluorescein tear break-up time (TBUT); and corneal-conjunctival staining, performed at baseline (T0), after 7 (T7) and 28 (T28). Schirmer -test, corneal endothelial cell count, intraocular pressure, and fundus examination were performed at T0 and T28. The main outcome measures were TBUT and corneal-conjunctival staining as markers of ocular surface homeostasis. For statistical analysis, Student’s -test and Wilcoxon test were used as appropriate. No significant alterations of the safety parameters were found throughout the study. Further, at T28 a significant improvement of burning, ocular dryness, foreign body sensation, and watery eyes (T0 vs. T28  < 0.03) were observed; corneal-conjunctival staining improved at T28 (T0 vs. T28  < 0.0001), and TBUT improved already at T7 (T0 vs. T7  = 0.0008) lasting so till the end of the study. The only adverse event was mild burning at instillation for the first 3 days of treatment in most of the patients. The treatment with 0.6% PVI was safe and well tolerated in a group of patients with a damaged ocular surface.