Products from aquaculture have sometimes been focused on the problems caused by the contamination of chemical agents as the use of chemical agents in aquaculture has been annually increased. The risk of contamination of products by chemical agents is greater in freshwater than in seawater. In order to evaluate the food safety of a fish grown in freshwater, we investigated the residues of antibiotics (tetracycline, oxolinic acid and ciplofloxacin) and malachite green in cultured rainbow trout, Oncorhynchus mykiss. Malachite green, which was prohibited in the application of aquaculture, was not detected in samples tested in this study. The residual content of tetracycline was determined to be less than the permissible amount, <0.2 mg/kg. The contents of ciplofloxacin was also less than the permissible amount, <0.1 mg/kg. However, in case of oxolinic acid, one of samples was only exhibited higher content than the permissible amount (<0.1 mg/kg). The results obtained in this study suggested that the control and regulation of chemical agents such as antibiotics was important to maintain a safe and worry-free seafood supply.