Nowadays, the urgent task of pharmacy and chemistry is to increase the bioavailability and effectiveness of medicines to overcome the resistance of pathogens. Nanoparticles based on biologically active substances have a special potential for solving this problem. The purpose of this work was the synthesis of nanoparticles of ethyl thiosulfanilate (ETS) with rhamnolipids (RL), establishment of physicochemical properties and antimicrobial action, comparison of the activity of nanoparticles and ETS-RL compositions. Using the methods of dynamic light scattering and scanning electron microscopy, it was established that aqueous solutions of the obtained ETS-RL compositions and nanoparticles formed spherical micelles, with hydrodynamic radii of 138 nm and 109 nm, respectively. The influence of the size of nanoparticles on their properties is shown, in particular, solutions of nanoparticles have a higher emulsifying and wetting ability compared to compositions. Solutions of ETS-RL compositions and nanoparticles show antimicrobial activity against Staphylococcus aureus, Micrococcus luteus, Escherichia coli, Alternaria alternata, Fusarium oxysporum, Candida tenuis. The obtained results testify to the effectiveness of the developed compositions and nanoparticles of ETS-RL and confirm the perspective of their use in pharmacy.