PURPOSE To investigate ocular bacterial count before and after antisepsis with aqueous chlorhexidine (AC) or povidone-iodine (PI) and to assess discomfort with each agent. METHODS Bacterial swabs were taken from participants' eyes before and after antisepsis. These underwent microscopy, culture, and sensitivity testing. Aqueous chlorhexidine drops were administered to left eyes and PI to right eyes. Participants rated their pain (scale 0-10) for each eye but were blinded to the type of drop. RESULTS There were 20 participants (17 women, 3 men), and the mean age was 43 years. Pain scores were significantly higher in right (PI) than in left (AC) eyes (mean 7 vs. mean 2, P < 0.001). No abnormalities were detected on specimen microscopy and gram staining. Seven preantisepsis swabs (three left and four right) grew bacteria in culture. Two postantisepsis swabs grew bacteria in primary culture plate (1 after AC and 1 after PI). For an additional one post-PI swab, bacteria were detected in enrichment broth only. CONCLUSION The efficacy of AC and PI are similar, and patient discomfort is lower with AC. Aqueous chlorhexidine is a good alternative to PI for antisepsis before intravitreal injection.