Purpose <p>To investigate the prevalence and spectrum of microbial contamination of donor corneoscleral rims and storage medium in keratoplasty and to report a case of donor-derived fungal keratitis.</p> Study design <p>Single-center, retrospective study.</p> Methods <p>We reviewed 285 eyes that underwent keratoplasty at Hiroshima University Hospital between October 2022 and January 2025. Donor corneas were preserved using Optisol^®-GS. The donor rim and storage medium were separately cultured intraoperatively, and the minimum inhibitory concentrations (MICs) were determined.</p> Results <p>Culture positivity was identified in 10 of the 285 eyes (3.5%): six from donor rims and four from storage media, with no eye yielding positive results in both. All tested bacterial isolates were Gram-positive; three of six tested were not susceptible to levofloxacin and gatifloxacin (MIC ≥4&#xa0;μg/mL), including methicillin-resistant <i>Staphylococcus aureus</i>, but retained susceptibility to vancomycin and meropenem. All fungal isolates were <i>Candida</i> with low MICs for voriconazole and micafungin. <i>C. albicans</i> and <i>C. glabrata</i> were recovered from the mate corneas of a single donor; the recipient of the cornea contaminated with <i>C. albicans</i> developed keratitis requiring repeat penetrating keratoplasty, whereas the recipient of the <i>C. glabrata-</i>contaminated cornea did not.</p> Conclusion <p>Adding storage medium to the rim culture improved the detection of donor-associated microorganisms. When donor-derived <i>C. albicans</i> keratitis developed, susceptibility data from contemporaneous cultures enabled prompt targeted antifungal therapy and globe preservation, although repeated penetrating keratoplasty was ultimately required.</p>