Aquaflor is an aquaculture feed premix containing 50% florfenicol and is approved for use in more than 50 countries to control mortality in a variety of cultured fishes caused by diseases associated with infectious bacterial pathogens. As part of an effort to expand the current approval in the United States, we conducted a study to evaluate the safety of Aquaflor to Yellow Perch Perca flavescens when administered in feed at 0× (0 mg/kg), 1× (15 mg/kg), 3× (45 mg/kg), or 5× (75 mg/kg) the proposed maximum therapeutic treatment dose of 15 mg florfenicol·kg fish−1·d−1 for 20 consecutive days, 2× the proposed therapeutic treatment duration of 10 consecutive days. Fingerling Yellow Perch (7.8 ± 1.6 cm and 5.0 ± 3.4 g; mean ± SD) were stocked into flow‐through test tanks at 15 fish per tank, and treatments were randomly assigned to tanks in triplicate. At the end of the 20‐d exposure period, mean cumulative mortality in the 0× and 3× groups (6.7% for both) was greater than that in the 1× and 5× groups (2.2% and 0.0%, respectively); however, differences among the groups were not significant (P = 0.3741). Throughout the study, general fish behavior was characterized as normal, and fish consumed virtually all feed offered. Fish health and histology assessments revealed no signs or lesions associated with toxicity of florfenicol. In conclusion, there is an adequate margin of safety associated with administering Aquaflor‐medicated feed to fingerling Yellow Perch at the proposed therapeutic treatment regimen of 15 mg florfenicol·kg fish−1·d−1 for 10 d.