Background: Laser is a novel physical therapy technique used to treat various conditions, including wound healing, inhibition of bacterial growth, and postoperative wounds. High-power pulsed alexandrite laser therapy is one of the most prevalent forms of laser therapy, which is a noninvasive method for treating various pathological conditions, thereby enhancing functional capacities and quality of life. It is a modern medical and physiotherapeutic technology. Generally, the Alexandrite laser emits infrared light with a wavelength of 755 nm, allowing it to propagate and penetrate tissues. Objective: This study focused on the application of a high-power pulsed alexandrite laser in vitro to evaluate the effect of a pulsed alexandrite laser on antibiotic-resistant bacteria utilizing varying exposure times, pulse durations, and laser fluencies to determine which dose is more effective on S. aureus bacteria. Method: The laser used in this study was the alexandrite laser which was considered a pulsed laser and had the following parameters: The wavelength was 755 nm, the beam diameter was (14 mm), the exposure times varied (30, 60, 90) seconds, the laser fluency (5, 10, 15 and 20 J.Cm-2). The study was carried out after the bacteria were diagnosed as being antibioticresistant. They were exposed to different doses of Alexandrite laser. Three samples of bacteria were exposed to laser beams for 30 seconds with a 5ms pulse duration and with a laser fluency of 5J/cm2, and this process was repeated with laser fluencies of 10, 15, and 20. This procedure was repeated using exposure times of 60sec and 90sec. As well as, this process was repeated by exposure with 30 sec, 60 sec and 90 sec exposure times, 10ms and 20ms pulse durations and with different laser fluencies 5, 10, 15 and 20J/cm2, separately. Results: A significant reduction (p = <0.0001) in the mean values of the colony was observed with the increase of laser fluency doses compared with control at the same pulse duration. A significant reduction (p = <0.0001) in the mean count of the colonies was observed in the comparison between two laser fluences at the same pulse duration. In conclusion, the exposure times, pulse durations and laser fluencies of pulsed alexandrite laser showed an effect on the mean count of the colonies of S aureus bacteria and determined the effective dose. Keywords: laser, Staphylococcus aureus, Bacteria growth