Rhamnolipids (RhLs) and polysaccharides (PS) produced by Pseudomonas aeruginosa are promising biosurfactants and bioactive compounds for agricultural and biotechnological applications. This study aimed to optimize RhL production by P. aeruginosa strain ONU 301 and evaluate the antiviral activity of its extracellular metabolites for plant protection. To improve the purification of RhLs from fermentation broth, a heterophase extraction method was developed, and its efficacy was confirmed by thin-layer chromatography. Among the tested metabolites, the PS fraction exhibited the highest antiviral activity, reducing tobacco mosaic virus (TMV) infectivity on Datrura stramonium leaves by 24–92% at concentrations ranging from 1 to 1000 μg/ml. The RhL fraction showed moderate activity, decreasing infection by 19 – 59% at higher concentrations (10 – 1000 μg/mL). No antiviral activity was observed in the low-molecular-weight fraction after removal of RhL and PS biopolymers. Both fractions were non-phytotoxic, supporting their agricultural potential. Based on the obtained results, PS and RhLs from P. aeruginosa ONU-301 are proposed as promising bio-based nanomaterials for the development of complex liposomal formulations with broad-spectrum applications, particularly in eco-friendly plant protection strategies. Future research will focus on the biological and physicochemical characterization of these metabolites and the investigation of their potential applications in biotechnology, supramolecular chemistry, and agriculture.