Traditionally, malachite green administrated as in-bath treatment was the most effective and common strategy used in freshwater aquaculture systems to control infections of the ciliate protozoan parasite Ichthyophthirius multifiliis Fouquet, 1876. After the ban of malachite green in the USA and Europe to be used in fish for human consumption, there has been extensive research destined to find efficacious replacements. Recently, peracetic acid-based compounds have demonstrated a strong cytotoxic effect in vitro and in vivo against I. multifiliis. In the present study, we tested the efficacy of a hydrogen peroxide, peracetic, acetic and peroctanoic acid-based formulation (HPPAPA) to eliminate the free-living stages of I. multifiliis (tomonts, cysts and theronts). The results obtained showed that the administration of low doses (8, 12 or 15 mg/l) of a specific HPPAPA-based product during a short window of exposure (60 min) kills nearly all free-living stages of I. multifiliis (theronts, tomonts and cysts) within the window of treatment (∼100% mortality for all the stages; one-way ANOVA, P ≤ 0.001). Of note, even the lowest concentration of HPPAPA tested (8 mg/l) was able to disrupt normal cyst development and therefore theront release. The demonstrated in vitro efficacy of the peracetic acid-based product tested on the present study suggests its great potential to control I. multifiliis infections in commercial aquacultural systems.