Laser ablation in liquid was utilized to prepare a TiO2 NP suspension in in deionized distilled water using Q-switch Nd: YAG laser at various laser energies and ablation times. The samples were characterized using UV–visible absorption spectra obtained with a UV–visible spectrophotometer (UV-Vis,) Fourier transform infrared (FTIR), X-ray diffraction (XRD), and transmission electron microscope (TEM). While, UV-Vis spectra showed the characteristic band-to-band absorption peak of TiO2 NPs in the UV range. FTIR analysis showed the existence of O-Ti-O bond. XRD patterns indicated the presence of (101) and (112) plane crystalline phases of TiO2. TEM images showed a spherical-like structure of TiO2 NPs with various size distributions depending on the ablation period. It was also found that there is a relationship between laser ablation time and TiO2 NP size distribution, where longer ablation times led to the smaller size distribution. The antibacterial activity of TiO2 NPs was evaluated with different species of bacteria such as Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, and Staphylococcus aureus, using the liquid approach. The optimum activity of TiO2 NPs is found to be against E. coli at 1000 μg mL−1. Furthermore, adding, TiO2 NPs (1000 μg mL−1) in the presence of amoxicillin has a synergic effect on E. coli and S. aureus growth, as measured by the well diffusion method. However, both E. coli (11.6 ± 0.57mm) and S. aureus (13.3 ± 0.57mm) were inhibited by this process.