Background: The main etiological agent for dental caries is Streptococcus mutans bacterial strain. Recently, metallic nanoparticles and diode laser have been introduced as a new method in bacterial decontamination. Nanoparticles have very small particles size that has greater surface area and exert their antimicrobial effect by interacting with the negatively charged bacterial cell wall, while lasers have become increasingly popular in dentistry due to their multiple uses, and one such important application is tooth disinfection. Objective: Present study was conducted to evaluate antimicrobial efficacy of silver (AgNPs) and gold nanoparticles (AuNPs) with and without diode laser (970 nm, 15 Hz, 100 mJ, 2 W, each cycle was 10 s and repeated 6 times with intervals of 5 s for each sample) irradiation against experimentally inoculated S. mutans bacteria in the samples. Material and methods: Thirty freshly a traumatically extracted premolars human tooth were collected, and cylindrical cavity in crowns with 4 mm diameter and 5 mm depth was prepared and inoculated with S. mutans for 24 h. The tooth were randomlly divided into equal expermintal groups (6 each): group 1, positive control group by S. mutans bacterial strain; group 2, irrigation with silver nanoparticles (AgNPs) for 3 min; group 3, irrigation with gold nanoparticles (AuNPs) for 3 min; group 4, 3 ml AgNPs + irradiation with diode laser for 60 s; and group 5, 3 ml AuNPs + irradiation with diode for 60 s. The specimens were collected from the teeth sample, and colony-forming units (CFU's) were observed by counting the bacteria colony on plates. Results: A significant difference was found among all the groups in comparison to the control group (p < 0.05). The greatest reduction in CFU's was observed in combination of AgNPs with diode laser group. Conclusions: The NPs in combination with diode laser irradiation has the potential to be used as dentine disinfectant.