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This study offers new insights into dysprosium oxide nanoparticles (nDy2O3) exposure on E. coli with respect to its metabolic activity and structural integrity. The toxicity of nDy2O3 was evaluated for two array-based techniques, Live/Dead and respirometric assays. Our work is novel since new nDy2O3 toxicity data was produced covering manufacturer recommendations for toxicity assessments, environmental and industrial nDy2O3 effluent concentrations and metabolic activity. This is highly relevant to the evaluation of the toxic effect of nanoparticles since the physicochemical properties of the nanoparticles can differ greatly depending of the physicochemical characteristics of the aqueous solution. With an understanding of the nanoparticles fate in aqueous media, a careful selection of appropriate toxicological methodologies can be made to improve the accuracy of future nanotoxicological studies.