The present study aimed to evaluate the antiparasitic efficacy (AE) of Nootkatone, a component of grapefruit oil, against the Argulus parasite infecting goldfish under both in vitro and in vivo conditions. Under in vitro conditions, AE of Nootkatone at concentrations of 60, 70, and 80 ppm against Argulus was found to be 33.3, 50, and 100%, respectively, within 6h. An estimated median effective concentration (6 h EC50) of Nootkatone for Argulus was found 65.61 ppm. An acute toxicity test of Nootkatone on goldfish revealed that the median lethal concentrations (LC50) of 23.60 ppm at 12 h, 19.23 ppm at 48 h, 18.36 ppm at 72 h, and 17.57 ppm at 96 h. Further, under in vivo testing, Nootkatone at 18.4 ppm resulted in the complete detachment of Argulus from the goldfish within 9 h of bath treatment. Additionally, the detached Argulus, when left in the same treatment solutions, exhibited complete parasite mortality after an additional 3 hours of exposure. The therapeutic index (TI) calculated for Nootkatone at 12 h was 1.54, indicating a relatively safe dose for treating argulosis in goldfish. Thus, the present findings suggest that Nootkatone can be used as a promising antiparasitic agent against Argulus in the ornamental fish industry.