Fatty acid hydroperoxides (HPOs) are amphiphilic molecules naturally produced by plants in stressed conditions and involved in plant immunity as signalling molecules. 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 work, the potential of two HPO forms, 13-HPOD and 13-HPOT, as plant defence elicitors and the underlying mechanism of action are investigated. Both HPOs trigger Arabidopsis innate immunity. They increase plant resistance to the pathogenic fungi Botrytis cinerea and activate early immunity-related defence responses, like ROS production. As our previous study has suggested that HPOs are able to interact with the plant plasma membrane (PPM) lipid fraction, 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. 13-HPOT, with an additional double bond compared to 13-HPOD, exerts a higher effect by fluidifying and reducing the thickness of the bilayer. Correlation between biological assays and biophysical analysis suggests that lipid amphiphilic elicitors that directly act on membrane lipids might trigger early plant defence events.