Acanthocephalosis is caused by the endoparasite Neoechynorhyncus buttnerae and affects fish farmed in the Amazon. This study assessed the efficacy of therapeutic levamisole hydrochloride (LVC) baths against N. buttnerae and its effects on juvenile tambaqui blood parameters. In vitro and in vivo tests were carried out, the latter employing two experimental therapeutic LVC bath protocols. Concerning in vitro efficacy, the T75 (75 mg.L−1 LVC) and T100 (mg.L−1 LVC) treatments were 100% effective in 15 min, while the T50 (50 mg.L−1 LVC) and T25 (25 mg.L−1 LVC) treatments required parasite exposure for 45 and 60 min, respectively. During exposure, the parasites displayed reduced motility, proboscis retraction, coiling into a spiral shape, body rigidity and swelling. The LVC LC50–72h for juvenile tambaqui was 115 mg.L−1. Regarding in vivo efficacy for Protocol I (8‐h bath), the T125 resulted in 82% effectiveness, while in Protocol II (two 8‐h baths with a 24‐h interval), the T115 treatment (115 mg.L−1 LVC) achieved 95.6% effectiveness without clinical intoxication signs, despite behavioural changes. No significant changes were observed in fish blood parameters. LVC was, therefore, highly effective both in vitro and in vivo in controlling the acanthocephalan N. buttnerae without compromising tambaqui juvenile homeostasis.