In this study, crystalline ZrO2 nanoparticles were synthesized in one pot at room temperature using an extremophilic Acinetobacter sp. KCSI1 and characterized using various techniques to study its structural, optical and crystalline properties. The average size of the ZrO2 nanoparticles was found to be 44 ± 7 nm. The XRD and Raman spectra showed the crystalline structure of ZrO2. HRTEM and SAED images showed well aligned ordered crystal lattice nanoparticles. The zeta potential of Bio-NP of ZrO2 has been found to be 36.5 ± 5.46 mV. The mechanical behaviour such as hardness and Young's modulus of Bio-ZrO2 NPs were determined using atomic force microscopy (AFM) and found to be 9.206 ± 2.22 and 0.285 ± 0.13 GPa, respectively. No significant cytotoxicity for ZrO2 nanoparticles was observed when tested with mouse fibroblast cells (L929), suggesting that the synthesized ZrO2 nanoparticle is biocompatible and safe for environmental applications. Copyright © 2019. Published by Elsevier B.V.