Root canal disinfection, rather than mere debridement, has become a central goal of endodontic treatment in recent years. To adequately discuss the possibility of microbial elimination, one has to have a good understanding of the methods available for microbial detection. This chapter discusses the common methods of detection of root canal microorganisms. To detect microbial presence in root canals, a sample has to be obtained and analyzed. As bacterial presence or persistence in the root canal is causative of apical periodontitis, and several studies have shown the close association between bacterial elimination and long-term outcomes, it follows that determination of the efficacy of root canal disinfection is a reasonable surrogate outcome. It is important to realize that the root canal system presents a variety of different clinical conditions and types that require different strategies for optimal disinfection. One of the most notable variables is the size of the root canal.