The aim of this study was to compare microbiologically the effects of the CO2 Laser and Chlorohexidine Gluconate (4%) on Staphylococcus aureus infected cutaneous wounds. Wound infection constitutes a big risk for patients and it is usually associated to increased morbidity, mortality and hospital costs. It is accepted that local treatment of these infections is effective. Standardized wounds created on the dorsum of 36 rats were infected with Staphylococcus aureus and treated during six days as follows: Group I: Chlorohexidine Gluconate applied to the wound surface during one minute during six days; Group II: Single CO2 Laser irradiation (8W,CW, unfocused, 8cm focal distance, 81530W/cm2), maintaining surface debris; Group III: Single CO2 Laser irradiation (8W,CW, unfocused, 8cm focal distance, 81530W/cm2), removing the surface debris. Daily samples were taken for microbiological analysis. Seven days after wounding the animals were killed a final sample taken. The use of Chlorohexidine Gluconate solution and the CO2 laser with the removal of the surface debris result in a significant reduction on the infectability of the Staphylococcus aureus when compared to non-treated infected wounds (p=0.00 e p=0.02). However, if the debris is left on the wound surface the resolution of the infection is less significant and results in non-significant differences on the number of Staphylococcus aureus when compared to non treated controls (p=0.14). No difference on infectability of the Staphylococcus aureus was detectable when the debris was removed of the surface of the wounds and when the Chlorohexidine solution was used (p=0.05). Therefore, the use of the CO2 laser would improve the resolution of the infection without further irradiating the tissue and consequently without further impairing wound healing. The fact that significant differences were observed between the two modalities of CO2 treatment indicates that the surface debris acts as a culture medium for bacterial growth keeping a higher infectability of the wound in which the debris was not removed (p=0.04).