Simple SummaryParasitic infections are a major problem in aquaculture, and there is a growing need for alternative treatments. This study investigated the efficacy of levamisole hydrochloride againstMicrocotyle sebastisinfections in Korean rockfish. In vitro experiments showed thatMicrocotyle sebastiswas sensitive to levamisole hydrochloride, and in vivo experiments tested different administration methods. In a controlled laboratory environment, oral administration has been demonstrated to exhibit efficacy without imposing any adverse effects on fish health. While these findings necessitate further investigation for practical application in actual aquaculture settings, they suggest the potential of levamisole hydrochloride as a therapeutic agent againstMicrocotyle sebastisinfections in Korean rockfish. Consequently, this may contribute to the improvement of parasitic control strategies in aquaculture.AbstractParasitic infections pose significant challenges in aquaculture, and the increasing resistance to conventional anthelmintics necessitates the exploration of alternative treatments. Levamisole hydrochloride (HCl) has demonstrated efficacy against monogenean infections in various fish species; however, research focused onMicrocotyle sebastisinfections in Korean rockfish (Sebastes schlegelii) remains limited. Therefore, this study aimed to evaluate the efficacy of levamisole HCl againstM. sebastisinfections in Korean rockfish with the goal of optimizing anthelmintic usage in aquaculture. In this study, we first assessed the susceptibility ofM. sebastisto levamisole HCl in vitro. Subsequently, in vivo evaluations were conducted to assess the drug’s efficacy, safety, and to identify optimal administration methods. In vitro experiments revealed concentration-dependent sensitivity ofM. sebastisto levamisole HCl, with a minimum effective concentration (MEC) of 100 mg/L. In vivo experiments employed oral administration, intraperitoneal injection, and immersion treatments based on the MEC. Oral administration proved to be a safe method, yielding efficacy rates of 27.3% and 41.6% for 100 mg/kg and 200 mg/kg doses, respectively, in contrast to the immersion and injection methods, which induced symptoms of abnormal swimming, vomiting, and death. Biochemical analyses conducted to assess the safety of levamisole HCl revealed a transient, statistically significant elevation in the levels of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) on day three post-administration at 20 °C. Following this, no substantial differences were observed. However, at 13 °C, the enzyme levels remained relatively consistent, emphasizing the role of water temperature conditions in influencing the action of levamisole HCl. Our research findings substantiate the efficacy of levamisole HCl againstM. sebastisin Korean rockfish, underscoring its potential for safe oral administration. These results provide valuable insights for developing parasite control strategies involving levamisole HCl in Korean rockfish populations while minimizing adverse impacts on fish health and the environment. However, this study bears limitations due to its controlled setting and narrow focus. Future research should expand on these findings by testing levamisole HCl in diverse environments, exploring different administration protocols, and examining wider temperature ranges.