Inorganic nanostructures are highly recognized for their potential use in the development of new functional materials for biomedical applications. In this study, we investigated the antibacterial efficiency of molybdenum trioxide (MoO3) nanoplates against four types of pathogenic bacteria. MoO3 nanoplates are synthesized by a simple wet chemical approach. X-ray diffraction and FT-IR analysis showed the presence of an orthorhombic phase of MoO3 nanoplates. Field emission scanning electron microscope studies confirmed the formation of plate-like structures of MoO3. The minimum inhibitory concentration (MIC) of MoO3 nanoplates against pathogenic bacteria was evaluated using a microdilution method. MICs such as 8 μg/mL (against Escherichia coli and Salmonella typhimurium), 16 μg/mL (against Enterococcus faecalis), and 8 μg/mL (against Bacillus subtilis) show that MoO3 nanoplates have predominant antibacterial activity compared to the standard antibiotic, kanamycin. Evaluation of bacterial enzymatic (β-d-galactosidase) activity in the hydrolysis of o-nitrophenol and β-d-galactopyranoside suggested the disruption of the bacterial cell wall mechanism responsible for bacterial toxicity.