The antimicrobial effects of engineered nanomaterials (ENMs) on the bacteria are poorly understood under anoxic conditions. Knowing about these effects can better inform environmental risk assessments. This study investigates the inhibitory effects on growth of silver nanoparticles at 3–6 mg l⁻¹, CuO nanoparticles at 100 mg l⁻¹ or CdTe quantum dots at 3 mg l⁻¹ towards Escherichia coli K-12 MG1655. These exposure concentrations reflect sub-lethal thresholds identified from a screening exposure in 96-well plates. Uncoated or functionalised ENM variants (negative carboxylate, positive ammonium and neutral polyethylene glycol) are investigated, along with bulk materials or metal salts. Bacterial growth is satisfactory, with test reproducibility of 11% coefficient of variation in oxic flasks, and 13% in anoxic serum bottles. Irrespective of the aeration conditions, Ag test materials cause most bacterial growth inhibition, followed by CdTe and CuO test materials. The bulk form of Ag, CuO and CdTe, as well as the Cu, Cd and Te metal salt, cause more growth inhibition under anoxic conditions, compared to oxic conditions (t-test, p < 0.05). However, by surface coating type, there is no clear trend in bacterial growth inhibition, and this also applies across all test materials. Under anoxia, the appearance of millimolar concentrations of fermentation products confirm that the organism can ferment glucose in the presence of ENMs, but with detrimental effects, as there is less energy available to biologically control the presence of metals. Hence overall, the ENMs are more toxic to E. coli under anoxic conditions, suggesting that regulatory tests with microbes that use oxic conditions may underestimate the hazards.