The effects of six metal oxide nanoparticles (MO-NPs) on the activity and biosynthesis of an enzyme (β-galactosidase) were examined using a mutant strain of E. coli. Different sensitivities were observed according to the type of NP and metabolic process. The toxic effects on enzyme activity were significantly greater than on biosynthesis (p < 0.011), except in the presence of NiO. In both cases, ZnO NP caused the greatest inhibition among the tested NPs, followed by CuO. The EC50s for ZnO were 0.19 and 3.68 mg/L for enzyme activity and biosynthesis, respectively. Similar orders of toxicity were observed as follows: ZnO > CuO > NiO > Co₃O₄ > TiO₂, Al₂O₃ for enzyme activity; and ZnO > CuO > NiO ≫ Al₂O₃, TiO₂, Co₃O₄ for the biosynthetic process. More systematic research, including in-depth studies like investigation of the molecular mechanisms, is necessary to elucidate the detailed mechanisms of inhibition involved in both metabolic processes.