Although zinc oxide (ZnO) nanoparticles (NPs) are recognized for their antimicrobial properties, little attention has been given to their laser ablation synthesis and antibacterial performance. ZnO NPs were prepared from ZnO metal in distilled water using 300 pulses of a Q-switched laser at variable energy levels (200–500 mJ). The effects of preparation conditions on the structural properties of ZnO NPs were analyzed. X-ray diffraction was used to evaluate the crystallographic structure and crystallite size of the samples, confirming a hexagonal structure consistent with zinc. The band gap of ZnO was found to decrease with increasing laser energy during pulsed laser ablation in liquid due to the increased nanoparticle size and the presence of crystal defects. Scanning electron microscopy images revealed s homogeneous morphology of the ZnO NPs, with diameters ranging from 19.6 to 36.58 nm. Atomic force microscopy analysis demonstrated that increasing laser energy leads to a larger grain size and a corresponding increase in surface roughness. Antibacterial activity of the prepared ZnO NPs was observed against Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative), confirmed through the agar well-diffusion assay. ZnO NPs prepared at 200 and 300 mJ laser energies exhibited significant inhibitory effects on the growth of all tested bacterial pathogens. This antibacterial activity is attributed to the NPs’ large surface area and enhanced surface energy due to their nanoscale size. Findings demonstrate the strong antibacterial potential of laser-synthesized ZnO NPs at low energies, offering a novel perspective on nanomaterial interactions with bacterial pathogens.