The present work highlights a low-cost, ecofriendly and simple procedure for the synthesis of zinc oxide nanoparticles (ZnONPs), mediated by extracellular secondary metabolite pyoverdine – a siderophore produced by Pseudomonas aeruginosa. The in vitro bio-reduction of zinc nitrate into ZnONPs in the presence of siderophore pyoverdine was ascertained by UV–visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy and transmission electron microscopy (TEM) analysis. The synthesized ZnONPs were distinguished by a characteristic absorption peak at 416 nm in the UV–visible range. The TEM pictures of ZnONPs showed pseudospherical morphology, with particle size ranging between 50 and 100 nm. X-ray diffraction peaks obtained at 31.73^°, 34.37^°, 36.24^°, 47.40^°, 56.68^°, 63.0^° and 67.8^° corresponding to the lattice plane of [100], [002], [101], [102], [110], [103] and [112] suggested the polycrystalline wurtzite structure of the nanoparticle. The ZnONPs were found to have an antibacterial effect against Gram-positive bacteria (Staphylococcus aureus and Bacillus sp.) and Gram-negative bacteria (Escherichia coli), and also showed a fungicidal effect against phytopathogenic fungi such as Rhizoctonia solani, Fusarium sp. and Penicillium sp. The present approach of using exogenously produced bacterial siderophore offers a unique in vitro green method of synthesizing ZnONPs, where a direct contact between the toxic metal and biological resource material can be avoided.