Purpose: To determine the spectrum of conjunctival flora and the antibiotic susceptibility profiles of patients undergoing cataract surgery at a Midwestern university. Design: Prospective in vitro laboratory investigation of a patient cohort. Methods: Conjunctival cultures were obtained from patients undergoing cataract surgery at a single ambulatory center on the day of surgery before the instillation of any ophthalmic medications. Isolates and antibiotic susceptibility profiles were identified using standard microbiological techniques. Results: A total of 183 eyes were cultured, yielding 225 isolates. Twenty-seven eyes (14.8%) showed no growth. Coagulase-negative staphylococci (CNS) were the most commonly isolated organisms (74.8%). Overall susceptibility was highest for gentamicin (94%), which was also true of the CNS isolates (95.0%). A total of 64.5% of CNS isolates were sensitive to ciprofloxacin; 30.1% of CNS isolates were resistant to ≥3 classes of antibiotics; 46.6% of CNS isolates were oxacillin-resistant, and they were more resistant to antibiotics than their oxacillin-sensitive counterparts (P <.001), including fluoroquinolones (P <.001). Among eyes with multiple CNS strains, 41.4% had different antibiotic susceptibility profiles even though they were the same species. Conclusions: Our cohort harbored organisms with similar rates of antibiotic resistance as elsewhere in the country, including oxacillin resistance; however, the rate of fluoroquinolone resistance was less than in other reports. A surprisingly large proportion of different CNS strains from the same eye harbored different antibiotic susceptibility profiles. Our in vitro results, along with those of other investigators, should prompt further dialogue regarding antibiotic of choice for perioperative surgical prophylaxis in ophthalmic surgery.