The lipopeptide SPH6 isolated from the bacterial strain Pseudomonas H6 has surfactant properties and shows a lethal effect on all external stages of the problematic gill parasitic ciliate Ichthyopthirius multifiliis. The mode of action has remained enigmatic, but in the present study we have elucidated the mechanisms. I. multifiliis tomonts were exposed to a range of SPH6 concentrations (from 2.5 to 160 µg/mL), until the total motility inhibition of cells, cytoplasm and cilia. The parasites were subsequently examined by histology, light and scanning electron microscopy and enzyme activity assays (19 enzymes). The surfactant did not interfere with enzyme reactivity at any concentration, but at SPH6 concentrations 40 µg/mL and above the parasite cells became de-ciliated. Perforations were observed in the plasma membrane, and their origin and function may be associated with surfactant damage of the cell membrane or areas for extrusome release. Additional killing mechanisms may be involved, because lower concentrations were lethal as well, although the effect on density of cilia was not as pronounced. The study suggests that one mode of action mechanism is the surfactant attachment to and penetration of the parasite plasma membrane followed by detachment of cilia.