In order to ensure predictable decontamination of the root canal system, chemo-mechanical preparation of the root canal space is sometimes supplemented with the use of intracanal medication. As microbial control of the root canal space is fundamental to the resolution of apical periodontitis, root canal disinfection strategies haven been researched intensively. The use of intracanal medication as a supplementary step to the chemo-mechanical preparation of the root canal space is one of them. Because of the costs and limitations of clinical research it is relevant and common practice to first evaluate alternative or new root canal disinfection modalities in laboratory studies. This involves the simulation of a root canal infection in a laboratory model, on which different disinfection strategies can be tested. When modelling the infected root canal, different levels of infection can be discriminated: suspended bacteria, microbial biofilms and infected dentine. This review describes the experimental models associated with these infection levels and critically appraises their value and methodological details. Suggestions for relevant research methods and experimental models are given, as well as some good practices for laboratory-based microbiological studies.