Surfactants can be used as nanoparticle stabilizing agents. However, since synthetic surfactants are not economically viable and environmentally friendly, biosurfactants are emerging as a green alternative for the synthesis and stabilization of nanoparticles. Nanoparticles have been applied in several areas of industry, such as the production of biomedical and therapeutic components, packaging coating, solar energy generation and transmission and distribution of electrical energy, among others. The aim of this study was to synthesize, in a simple and green way, silver nanoparticles (AgNPs) using the biosurfactant produced by UCP 0899 as a stabilizer. AgNPs were examined and morphologically characterized using the techniques of ultraviolet-visible spectroscopy (UV-visible), scanning electron microscopy (SEM), zeta potential and energy dispersive X-ray spectroscopy (EDS). Newly formed silver nanoparticles showed a maximum UV-visible absorption peak at 400 nm, while a shift to 410 nm was observed in those stored for 120 days. SEM micrograph confirmed the formation of nanoparticles with an average size of 20 nm and with a predominant spherical structure, while a zeta potential of -60 mV suggested that the use of the biosurfactant promoted their stability. Stabilized nanoparticles were tested for their antimicrobial activity against bacterial isolates of , , and sp., as well as fungal isolates of and . At a concentration of 16.50 µg/mL, AgNPs inhibited the growth of all target microorganisms according to the following decreasing order: (95%), , (90%), (85%), sp. (75%) and (71%). These results suggest the potential use of the biosurfactant as a stabilizer of silver nanoparticles as an antimicrobial agent in different industrial sectors. Furthermore, the in vivo toxicity potential of biosurfactants was evaluated using the model. The larvae were treated with concentrations in the range of 2.5, 5.0 and 10 g/L, and no mortality was observed within the 24 to 72 h period, demonstrating non-toxicity within the tested concentration range. These findings support the safety, efficacy and non-toxicity of biosurfactant-stabilized nanoparticles.