Silver nanoparticles (Ag NPs) were prepared by laser ablation of a silver bulk immersed in cetrimonium bromide (CTAB). The immersed sheet was focused by a pulsed Nd:YAG laser at 1064 nm, 600 mJ at room temperature, a pulse duration of 10 ns, and a repetition rate of 5 Hz. The Ag NPs suspension was analyzed using UV–Vis spectroscopy, zeta potential (ZP), the Fourier transform infrared (FTIR), X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM). The peak at 425 nm was observed in the absorption spectrum of Ag NPs colloidal solution.Ag NPs' colloidal results are 20 mV for positively charged CTAB. The XRD pattern of the NPs revealed the presence of Ag phase planes (111), (200), and (220). All samples show aggregation-induced spherical nanostructures and large particles in FESEM images. The TEM images revealed nearly spherical NPs, with sizes ranging from 5–70 nm, for Ag NPs prepared with CTAB. The Ag NPs with concentrations of (0.0, 25, 50, 100, and 200) µg/mL were tested for antibacterial activity against a clinical isolate of Streptococcus mutans, and the concentrations of (0.0, 50, 75, 100, and 200) µg/mL were tested for in vitro hemocompatibility effects by measuring the hemolysis rate of red blood cells. Ag NPs' antibacterial activity against Streptococcus mutans was size and concentration-dependent, and the hemolysis rate showed low effects at low concentrations, so Ag NPs could be employed to treat and prevent dental caries, but to transform this technology into therapeutic and preventive measures, more investigation and development are required.