Bacteria that persist after endodontic disinfection procedures may lead to treatment failure. Over 50% of the bacteria found in endodontic infections are as-yet-uncultivated so investigations of bacteria that endure treatment procedures should include techniques that side-step cultivation. This culture-independent study evaluated the bacterial reduction promoted by intracanal disinfection procedures and identified the taxa persisting after treatment. Samples taken from the infected canals of teeth with apical periodontitis before treatment (S1), after instrumentation using NaOCl as irrigant (S2) and after interappointment medication with a calcium hydroxide paste (S3) were subjected to 16S rRNA gene clone library and real-time polymerase chain reaction analyses. The S2 and S3 samples from five of the 15 canals showed negative results. In the other cases, instrumentation and instrumentation/medication promoted a significant reduction (99.67% and 99.85%, respectively) in the number of bacteria when compared to S1 samples. Forty-three distinct bacterial taxa were identified, of which 24 (56%) were as-yet-uncultivated phylotypes. Nineteen of these 43 taxa (including eight as-yet-uncultivated phylotypes) were disclosed in post-treatment samples, with streptococci being the most prevalent taxa. Findings demonstrated that culture-independent methods provide a detailed insight into the effects of intracanal disinfection protocols, helping to define more effective strategies to deal with endodontic bacteria, including as-yet-uncultivated phylotypes.