ObjectiveTo compare gatifloxacin resistance in a population of ophthalmology patients who had received intravitreal injections (IVIs) with prophylactic topical gatifloxacin use to resistance in a similar population of patients who had not received IVI. DesignNested case–control study. ParticipantsFifty eyes of 50 patients who received prior IVI were enrolled, as were 50 control eyes. MethodsEach patient had a conjunctival swab performed on the study eye, which underwent microbial identification and testing for gatifloxacin resistance using the ellipsoid test to determine a minimum inhibitory concentration (MIC) value for each isolate. The primary outcome was susceptibility to gatifloxacin, as measured by the MIC of each isolate. ResultsA total of 111 bacterial isolates were obtained from 60 eyes; the remainder was culture negative. There were no significant differences in bacterial species or culture positivity rate between case and control eyes (50% in cases vs. 66% in controls, p = 0.16). The most common organism was coagulase-negative staphylococcus, comprising 64% of all isolates. Resistance to gatifloxacin was observed in 76% of the bacterial isolates and 38% of patients in the case group, as compared with 3% of bacterial isolates and 4% of patients in the control group, a result that was statistically significant (p = 0.0002 and 0.0008, respectively). The mean gatifloxacin MIC was also significantly higher in the case group. ConclusionsTopical gatifloxacin prophylaxis in those who receive IVI is associated with an increased rate of gatifloxacin resistance among conjunctival isolates.