---

The biosynthesis of nanoparticles (NPs) has been proposed due to its fast, clean, safe, and cost-effective production and being efficient alternative to conventional physicochemical methods. This study aimed to isolate and identify aquatic yeast strains for their potential to form Zinc oxide nanoparticles (ZnONPs). A yeast strain, NS02, with high tolerance against zinc ion (5.25 mM) was isolated using the enrichment technique and was selected as efficient candidate for the biosynthesis of ZnONPs under cell-free extract (CFE) strategy. The preliminary evaluation on the formation of ZnONPs was performed by visual observation and UV-visible absorption spectra of the biosynthesized ZnONPs. The morphology, size and elemental distribution of the nanoparticles were determined by Field emission scanning electron microscopy (FESEM) equipped with energy-dispersive X-ray (EDX). X-ray diffractometer (XRD) was used to identify the crystalline phase of the ZnONPs. Antibacterial activity of ZnONPs against pathogenic bacteria isolated from the clinical specimens was investigated using agar well diffusion method. The isolate NS02 was characterized based on their morphological properties and amplification the ITS-5.8S-ITS2 rDNA regions. The present study pioneered the capabilities of the native aquatic strain Rhodotorula pacifica for the extracellular synthesis of ZnONPs with CFE strategy. The biosynthesized ZnONPs had a growth inhibitory effect all tested clinical isolates due to their nanometric size and well-defined dispersity. This investigation is attempted to indicate the novel microbial sources of aquatic yeasts as biological plant in the synthesis of ZnONPs with antimicrobial activity under cell-free extract strategy.