During the last several years, various chemical methods have been used for synthesis of a variety of metal nanoparticles. Most of these methods pose severe environmental problems and biological risks; therefore the present study reports a biological route for synthesis of zinc oxide nanoparticles using Pseudomonas aeruginosa rhamnolipids (RLs) (denoted as RL@ZnO) and their antioxidant property. Formation of stable RL@ZnO nanoparticles gave mostly spherical particles with a particle size ranging from 35 to 80 nm. The RL@ZnO nanoparticles were characterized by UV-visible (UV-vis) spectroscopy, scanning electron microscopy, transmission electron microscopy, dynamic light scattering, Fourier transform infrared spectroscopy, X-ray diffraction (XRD), and thermal gravimetric analysis. The UV-vis spectra presented a characteristic absorbance peak at ∼ 360 nm for synthesized RL@ZnO nanoparticles. The XRD spectrum showed that RL@ZnO nanoparticles are crystalline in nature and have typical wurtzite type polycrystals. Antioxidant potential of RL@ZnO nanoparticles was assessed through 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl, and superoxide anion free radicals with varying concentration and time of the storage up to 15 months, while it was found to decline in bare ZnO nanoparticles. Similarly, the inhibitory effects on β-carotene oxidation and lipid peroxidation were also observed. These results elucidate the significance of P. aeruginosa RL as effective stabilizing agents to develop surface protective ZnO nanoparticles, which can be used as promising antioxidants in biological system.