Enrofloxacin is known to cause retinal toxicity in domestic cats. The hallmark lesion of enrofloxacin-associated retinal toxicity in domestic cats is thinning of the outer nuclear layer of the retina. Enrofloxacin is commonly used to treat bacterial infections in nondomestic felids because of its action against a wide spectrum of bacteria and the ability for it to be given orally. No previous studies have investigated the potential retinal toxicity of enrofloxacin in nondomestic felids. This retrospective study evaluated 81 eyes from 14 lions (Panthera leo) and 33 tigers (Panthera tigris) that had been enucleated or collected postmortem. The thickness of the outer nuclear retina was assessed in two separate sites in each eye by counting the rows of nuclei and by using digital image analysis software to determine the area of the nuclei at each site. Medical records were reviewed to determine the enrofloxacin dose for each cat. Cats that had not received enrofloxacin (n = 11) were compared with treated animals (n = 36). The outer nuclear layer thickness or area in treated versus untreated cats was not significantly different. Additionally, no clinical blindness was reported in any of the cats. This study showed no evidence of enrofloxacin-associated thinning of the outer nuclear layer in the lions and tigers evaluated, suggesting that enrofloxacin can be used safely in these animals.