The biosynthesis of Au-Pd core-shell nanoparticles (NPs) with wild-type Escherichia coli (Au-Pd/E. coli) is an excellent newly established, environmentally friendly synthetic method for the fabrication of nanomaterials compared to traditional chemosynthesis. However, there is insufficient detailed bioinformation on the compatibility, metabolic process, and mechanism of this approach. Metabolomics approaches have provided an excellent alternative to numerous bioinformatics approaches for shedding light on the biological response of an organism exposed to external stimuli at the molecular level. In this study, two different doses (8 and 80 μg/mL) of Au-Pd/E. coli were applied to treat human umbilical vein endothelial cells (HUVECs). Gas chromatography/mass spectrometry coupled with bioinformatics was used to analyze the changes in the HUVEC metabolome after treatment. The results indicated the occurrence of nonsignificant acute cytotoxicity based on cell proliferation and apoptosis analysis, while high concentrations (80 μg/mL) of Au-Pd/E. coli induced dramatic changes in energy metabolism, revealing a notable inhibition of the tricarboxylic acid (TCA) cycle along with the enhancement of glycolysis, the pentose phosphate pathway, fatty acid biosynthesis, and lipid accumulation, which was correlated with mitochondrial dysfunction. The metabolomics results obtained for this novel Au-Pd/E. coli-cell system could broaden our knowledge of the biological effect of Au-Pd/E. coli and possibly reveal material modifications and technological innovations.