The microbial aetiology of endodontic disease has been long established. The endodontic niche represents a very specific environment for the development of resilient bacterial community in the form of biofilms. The clinician should gather all the clinical evidence to achieve a thorough diagnosis of the type and level of infection present. In particular, those clinical signs that are associated with refractory endodontic infection should be detected. The systematic condition of the patient may lead to relapse of the endodontic pathology. However, local factors and signs are more preponderant indicating the presence of a refractory endodontic infection. The presence of long-standing fistulae, with associated development of extra-radicular biofilm or complex tooth anatomy will limit the possibility to fully disinfect the root canal system. The restorability and the coronal seal play an important role in preventing the risk of reinfection following root canal treatment. Also, several operative approaches are described to limit the chance of iatrogenic contamination of the root canal system. Particular emphasis is given to the more efficient shaping approaches. The irrigation still plays an ultimate role in the eradication of the endodontic biofilms. The correct irrigation regimen should be adopted to maximise its efficiency. Modern adjunct disinfection techniques are nowadays available: sonic and ultrasonic activation and photodynamic therapy are advanced tool to reach otherwise inaccessible areas. In the future, together with the aforementioned strategies, the detection in real time of the residual bacterial contamination post-treatment will help the clinicians to maximise the endodontic outcome.