Molybdenum disulfide, a type of two-dimensional layered material with unique properties, has been widely used in many fields. However, an exact understanding of its toxicity remains elusive, let alone its effects on the environmental microbial community. In this study, we utilized metabolomics technology to explore the effects of different concentrations of molybdenum disulfide nanosheets on Escherichia coli for the first time. The results showed that with increasing concentration of molybdenum disulfide nanosheets, the survival rate of Escherichia coli was decreased and the release of lactic dehydrogenase was increased. At the same time, intracellular concentrations of reactive oxygen species were dramatically increased. In addition, metabolomics analysis showed that high concentrations of molybdenum disulfide nanosheets (100, 1000 μg/mL) could significantly affect the metabolic profile of Escherichia coli, including glycine, serine and threonine metabolism, protein biosynthesis, urea cycle and pyruvate metabolism. These results will be beneficial for molybdenum disulfide toxicity assessment and further applications.