Introduction: Periapical lesions arise as a result of the activation and interaction of the host immune responses against root canal infection. Recently identified Toll-like receptors (TLR) seem to be involved in the recognition and development of immune responses against a myriad of microorganisms. However, very little information is available on the role of TLR in the induction of periapical lesions. Method: The role of TLR-2 and TLR-4 in the activation of murine macrophages stimulated using Fusobacterium nucleatum and Peptostreptococcus anaerobius was investigated. The production of nitric oxide (NO) and reactive oxygen species (ROS) was assessed. Results: The results demonstrate that TLR-2 and TLR-4 are involved in the production of ROS by activated macrophages. The microorganisms induced similar levels of NO production by TLR-2-competent and TLR-2-deficient macrophages, regardless of the addition of interferon-gamma (IFN-gamma), ruling out a role for TLR-2 in the NO production induced by these bacteria. Only P. anaerobius induced NO production by TLR-4-competent macrophages without the addition of IFN-gamma. However, after IFN-gamma addition, F. nucleatum induced macrophage NO production. Therefore, NO production stimulated by IFN-gamma and these microorganisms seems to be TLR-4-independent. Conclusion: TLR-2 seems to be involved in the induction of ROS production by macrophages in response to prevalent root canal bacteria, while only F. nucleatum induced ROS production by TLR-4-competent macrophages. Both microorganisms significantly induced large amounts of NO independent of TLR-2 and TLR-4. We conclude that microorganisms may participate in the induction and progression of periapical lesions through NO and ROS production by activated macrophages