Background: Intravitreal injections (IVIs) are considered one of the most important and minimally invasive procedures for treatment of many retinal diseases. Recently the instillation of povidone iodine to the surface of the eye is the only proven prophylactic measure that can lower the risk of endophthalmitis following intravitreal injection. Objective: The study aims to assess the antimicrobial efficacy of lower povidone iodine concentrations 2.5% and 1.25% in intravitreal injection. Methodology: This is a prospective interventional study that was conducted on 60 eyes of 45 patients with diabetic macular edema, retinal vein occlusion (RVO) and age related macular degeneration (AMD). Patients were divided into three groups, group one involved 20 eyes that received application of povidone iodine 5%, groups 2 and 3 received povidone iodine 2.5% and 1.25% respectively. Conjunctival swabs were taken before and after povidone iodine in the three groups, colony forming units (CFU) were calculated to detect the efficacy of povidone iodine in decreasing the bacterial load of the conjunctiva. The patient discomfort was recorded in a chart scale to evaluate pain. Results: The CFU was markedly decreased in the 3 groups with no detected statistical significance between all groups confirming the antiseptic efficacy of lower concentrations of povidone iodine. The lower concentration 1.25% showed more patient comfort and tolerability with lower score on pain chart scale. Conclusion: Lower concentrations of povidone iodine can achieve similar antiseptic effect compared to the standard 5% concentration with better tolerability.