Pulp regeneration is considered in cases where the dental pulp has been destroyed because of microbial irritation. Diverse oral and food-borne micro-organisms are able to invade the pulp space, form biofilm on canal walls, and infiltrate dentinal tubules. Prior to pulp regeneration procedures, the pulp space and dentinal walls need to be sufficiently disinfected to allow for and promote regeneration. The necessary level of disinfection is likely higher than that accepted for traditional endodontic therapy, because in traditional techniques the mere lowering of bacterial loads and prevention of bacterial access to periapical tissues is conducive to healing. Moreover, several of the non-specific antimicrobials used in traditional endodontic therapy may cause significant changes in remaining dentin that interfere with its inherent potential to mediate regeneration. Non-specific antimicrobials also suppress all microbial taxa, which may allow residual virulent micro-organisms to preferentially repopulate the pulp space. Therefore, it is important for endodontic pathogens to be studied by molecular methods that allow for a broad depth of coverage. It is then essential to determine the most effective protocols to disinfect the pulp space, with minimal disruption of remaining dentin. These protocols include the topical use of effective antibiotics, including newer agents that have demonstrated efficacy against endodontic pathogens.