Abstract The current study compares the antibacterial activity of zinc oxide nanostructures (neZnO). For this purpose, two bacterial strains, Escherichia coli (ATCC 4157) and Staphylococcus aureus (ATCC 29213) were challenged in room light conditions with the aforementioned materials. Colloidal and hydrothermal methods were used to obtain the quasi‐round and quasi‐diamond platelet‐shape nanostructures. Thus, the oxygen vacancy (V O ) effects on the surface of neZnO are also considered to assess its effects on antibacterial activity. The neZnO characterization was achieved by X‐ray diffraction (XRD), a selected area electron diffraction (SAED) and Raman spectroscopy. The microstructural effects were monitored by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Furthermore, optical absorption ultraviolet visible spectrophotometry (UV‐Vis) and X‐ray photoelectron spectroscopy (XPS) analyses complement the physical characterization of these nanostructures; neZnO caused 50 % inhibition (IC 50 ) at concentrations from 0.064 to 0.072 mg/mL for S. aureus and from 0.083 to 0.104 mg/mL for E. coli , indicating an increase in activity against S. aureus compared to E. coli . Consequently, quasi‐diamond platelet‐shaped nanostructures (average particle size of 377.6±10 nm) showed enhanced antibacterial activity compared to quasi‐round agglomerated particles (average size of 442.8±12 nm), regardless of Vo presence or absence.