Abstract: The dynamics of root canal system infections has been studied along the years. It has been established that bacteria and their by-products present in root canals are directly related to the development and maintenance of periapical lesion. The research to date found that a mixed population of facultative and obligate anaerobes as one of the aetiologic agents for periapical pathogenesis. With the possibility that resistant profiles may be emerging, it is prudent to characterize the antibiotic susceptibility pattern of these microorganisms involved in the periapical lesion of root canals. Thus, the objectives of this study are to isolate and identify the types of anaerobes present in the asymptomatic untreated root canal associated with periapical lesion. Its aim is also to determine the antibiotic susceptibility and resistant pattern of the isolated anaerobes. Therefore, the research methodology involved bacterial sampling, isolation and identification of the anaerobes from root canals associated with periapical lesion. The antibiotic susceptibility Kirby-Bauer test was carried out with Ampicillin (10 μg), Azithromycin (15 μg), Augmentin (30 μg), Metronidazole (5 μg) and Amoxicillin (10 μg). In addition, the antibiotic Minimal Inhibitory Concentration (MIC) of all five antibiotics was also carried out. The study has successfully isolated Actinomyces naeslundii, Prevotella intermedia, Porphyromonas asaccharolytica and Peptostreptococcus micros. It appears that metronidazole was 100% effective towards all isolated species. Azitromycin and Augmentin have exhibited high antibacterial effect (67%) towards the isolated anaerobes. Ampicillin and amoxicillin were the two least effective (40%) compared to the other three tested antibiotics in this study. This study has obtained two antibiogroup resistant patterns namely A_mp A_mox and A_mp A_zith A_ug A_mox from the isolated anaerobes of the root canal associated with periapical lesion in Malaysia.