Purpose: We sought to determine changes in the conjunctival bacterial flora and antibiotic resistance after topical antibiotic drops for infection prophylaxis were administered following intravitreal injections in patients with type 2 diabetes. Methods: This prospective and nonrandomized cohort study included 116 eyes of 116 treatment-naive patients with type 2 diabetes who received six serial intravitreal anti-vascular endothelial growth factor injections for macular edema. Three conjunctival cultures were obtained from each eye over the course of the study (Culture 1, baseline; Culture 2, 1 month after the third injection; and Culture 3, 1 month after the sixth injection). The study subjects were given topical moxifloxacin hydrochloride for 4 days after each monthly intravitreal injection. The growth patterns of conjunctival bacterial flora and the antibiotic resistance to several commonly used antibiotics were examined. Results: The rate of culture positivity increased significantly during the observation period (Culture 1, n = 47, 40.5%; Culture 2, n = 58, 50%; Culture 3, n = 76, 65.5%, p < 0.001). The bacterium with the highest baseline culture positivity was Staphylococcus epidermidis (n = 45, 38.8%), which increased significantly during the observation period (p < 0.001). No significant increase was noted in the culture positivity of the other bacteria with baseline culture positivity (p > 0.05). Regarding antibiotic susceptibility, significant increases in resistance to the fluoroquinolone group of drugs were noted (p < 0.001). No significant changes in sensitivity were detected in the other 11 investigated antibiotics that are commonly used in clinical practice (p > 0.05). Conclusions The use of topical moxifloxacin after each intravitreal injection significantly increases the fluoroquinolone resistance of the ocular surface flora and the culture-positivity rate of S. epidermidis in patients with type 2 diabetes.