Oxylipins are lipid‐derived molecules that are ubiquitous in eukaryotes and whose functions in plant physiology have been widely reported. They appear to play a major role in plant immunity by orchestrating reactive oxygen species (ROS) and hormone‐dependent signalling pathways. The present work focuses on the specific case of fatty acid hydroperoxides (HPOs). Although some studies report their potential use as exogenous biocontrol agents for plant protection, evaluation of their efficiency in planta is lacking and no information is available about their mechanism of action. In this study, the potential of 13(S)‐hydroperoxy‐(9Z, 11E)‐octadecadienoic acid (13‐HPOD) and 13(S)‐hydroperoxy‐(9Z, 11E, 15Z)‐octadecatrienoic acid (13‐HPOT), as plant defence elicitors and the underlying mechanism of action is investigated. Arabidopsis thaliana leaf resistance to Botrytis cinerea was observed after root application with HPOs. They also activate early immunity‐related defence responses, like ROS. As previous studies have demonstrated their ability to interact with plant plasma membranes (PPM), we have further investigated the effects of HPOs on biomimetic PPM structure using complementary biophysics tools. Results show that HPO insertion into PPM impacts its global structure without solubilizing it. The relationship between biological assays and biophysical analysis suggests that lipid amphiphilic elicitors that directly act on membrane lipids might trigger early plant defence events.