Copper oxide nanoparticles (CuO NPs) were synthesised with laser ablation of a copper sheet immersed in deionized water (DW), cetrimonium bromide (CTAB), and sodium dodecyl sulphate (SDS), respectively. The target was irradiated with a pulsed Nd: YAG laser at 1064 nm, 600 mJ, a pulse duration of 10 ns, and a repetition rate of 5 Hz. The CuO NPs colloidal were analyzed using UV-Vis spectroscopy, the Fourier transform infrared (FTIR) spectrometer, zeta potential (ZP), X-ray diffraction (XRD), transmission electron microscope (TEM) and field emission scanning electron microscopy (FESEM). The absorption spectra of CuO NPs colloidal showed peaks at 214, 215 and 220 nm and low-intensity peaks at 645, 650 and 680 nm for SDS, CTAB and DW, respectively. CuO NPs' colloidal results are (−21.6, 1.2, and 80 mV) for negatively, neutrally, and positively charged SDS, DW, and CTAB, respectively. The XRD pattern of the NPs revealed the presence of CuO phase planes (110) ( 111), (20-2) and (11-1). The TEM images revealed nearly spherical NPs, with sizes ranging from 10-90, 10-50, and 10-210 nm for CuO NPs mixed with DW, SDS and CTAB, respectively. FESEM images of all the synthesized samples illustrate the formation of spherical nanostructure and large particles are observable. The CuO NPs were tested for antibacterial activity against Streptococcus mutans by using the well diffusion method. In this method, CuO NPs prepared in DW at a concentration of 200 μg/mL showed a greater inhibition zone against Streptococcus mutans.