Tissue concentrations of the fluoroquinolone antimicrobial enrofloxacin and its active metabolite ciprofloxacin were determined over a 24 h period after a single topical application in the coqui frog (Eleutherodactylus coqui). Twelve frogs, scheduled for euthanasia unrelated to the study (due to exposure to a potentially lethal ranavirus), were randomly divided into four groups of three animals. Three groups received one dose of enrofloxacin topically (10 mg/kg), and the remaining group served as an untreated control. One group was euthanized and tissues (liver, kidneys) were collected at each of the following time points: 0 (control), 6, 12, and 24 h posttreatment. Tissues were pooled within each group, and concentrations of enrofloxacin and ciprofloxacin were measured using high-pressure liquid chromatography. Enrofloxacin tissue concentrations ranged from 0.2 to 0.44 μg/g, whereas ciprofloxacin concentrations ranged from 0.42 to 0.81 μg/g. Wildlife Conservation Society amphibian microbiology records from 2000 to 2010 were reviewed, and bacterial isolates and minimum inhibitory concentrations were determined. This study demonstrates that enrofloxacin is absorbed systemically after topical administration to coqui frogs, converts to ciprofloxacin, and results in measurable tissue concentrations for at least 24 h.