To investigate the effect of repeated povidone-iodine (PVI) application on the ocular surface parameters of patients who received intravitreal injections. In this prospective study, 52 eyes of 52 patients with age-related macular degeneration who underwent unilateral intravitreal injection at least three times in the last 1 year (intravitreal injection [IVI] group), 52 fellow eyes with no previous intravitreal injection (NIVI group), and 51 eyes of 51 healthy subjects (control) were included. Tear break-up time (TBUT), the Schirmer test, the Oxford staining score, the Ocular Surface Disease Index questionnaire, conjunctival impression cytology, and tear inflammatory cytokine levels (interleukin [IL]-1β and IL-6) were analyzed in all participants. The IVI group had lower TBUT and higher Oxford staining score than the NIVI and control groups ( P <0.05). No significant difference was found between the groups in the Schirmer test ( P =0.161). Conjunctival impression cytology analysis revealed that the IVI group had a significantly lower goblet cell count and significantly higher Nelson staging result than the NIVI and control groups ( P <0.05). As a result of tear cytokine analysis, although IVI and NIVI groups had higher IL-1β and IL-6 levels than the control group ( P <0.05), there was no difference between NIVI and IVI groups ( P ≥0.05). Repeated PVI application caused cytotoxic injury to the ocular surface, resulting in goblet cell loss and squamous metaplasia of epithelial cells. As a result, the stability of the tear film layer was found to be impaired and ocular surface-related symptoms developed in patients.