Herein, we used different methods, including metabolomics, to systematically investigate the influence of MSNs on E. coli. Our experimental results showed that high concentrations (100 μg/mL and more) of MSNs caused damage to cell membranes, induced ROS accumulation, and reduced viability. Exposure to low concentrations of MSNs (1, 10 μg/mL) on the other hand, increased the intracellular concentrations of many metabolites in E. coli. Interestingly, exposure to high concentrations of MSNs (100, 1000 μg/mL), conversely, lowered the concentrations of these same metabolites. Metabolomics analysis further revealed that exposure to high concentrations of MSNs could significantly affect several metabolic pathways such as amino acid related metabolism and pyruvate metabolism. These findings provide new insights for assessing MoS2 microbial toxicity.