Extensive use of nanomaterials in consumer products has invoked the concerns about interactions of nanoparticles with living organisms (including microorganisms). Zinc oxide nanoparticles (ZnO NPs) are well known for their antibacterial effect due to reactive oxygen species (ROS) generation. Therefore, their release into the environment is expected to raise major concern towards ecotoxicity. In the present study, we have studied the toxic effect of ZnO NPs on Deinococcus radiodurans, which is well known to show extraordinary resistant from the damaging effects of radiation. Result showed that ZnO NPs are significantly internalized into the bacterial cells and induce concentration-dependent toxicity with membrane damage. Genotoxicity studies revealed that ZnO exposure induces significant DNA damage to bacterial cells. All the observations evidenced that ZnO NPs induce significant ROS generation, protein oxidation and DNA damage with concomitant thiol depletion. Further, gene expression analysis showed that several DNA repair genes and metabolic pathway-related genes are downregulated upon ZnO NP exposure, with simultaneous increase in the expression of DNA damage response genes. Thus, the present study on toxicity of ZnO NPs on a model organism, D. radiodurans, inflicts the possible mechanism behind ZnO NP-mediated toxic effects on various other microbial organisms.