Dental caries continues to be a significant public health problem affecting mankind in many parts of the world. Microbial activities include the progressive localised destruction of teeth that without treatment, would eventually result in infection of the dental pulp and surrounding periapical tissues. Although the bacteria responsible for caries initiation and early caries progression have been extensively studied, the microbiology of dentine caries is reported to show considerable diversity and has not yet been fully identified. Few studies have analysed the microbiology of deep caries or examined the relationship between the microflora and the histopathy of chronic pulpits in symptomatic teeth. Matched carious dentine samples and dental pulps were obtained from teeth without evidence of periodontal disease but with coronal caries and symptoms of pulpits. Bacteria were cultured from the carious dentine samples under both anaerobic and microaerophilic conditions. Real-time polymerase chain reaction (PCR) technology was also used to identify and enumerate the bacteria. Development of the techniques for the efficient extraction of bacterial DNA from both Gram-negative and Gram-positive bacteria found in carious dentine was an essential prerequisite for molecular analysis. In addition, the dental pulps were processed and categorised into one of four groups on the basis of dominant pathology of the tissue (minimal inflammation, soft tissue degeneration, hard tissue degeneration, inflammatory degeneration). Analysis of the culture data indicated a predominance of Gram-positive bacteria, particularly lactobacilli, while Gram-negative bacteria were also present in significant numbers with Prevotella species the most numerous anaerobic group cultured. Real-time PCR indicated a greater anaerobic microbial load than that determined by colony counting. The total number of anaerobes detected by PCR was 41-fold greater, while Prevotella spp. and Fusobacterium ssp. were 82-fold and 2.4-fold greater respectively. PCR also identified the presence of Micromonas micros, Porphyromonas endodontalis and Porphyromonas gingivalis in 71%, 60% and 52% of carious dentine samples, respectively. Correlation matrices from the real-time PCR data revealed significant multiple associations involving Fusobacterium spp. in combination with P. endodontalis, M. micros and/or Prevotella in the tissue response categories of minimal inflammation, soft and hard disuse degeneration. A positive correlation was also observed between M. micros and P. endodontalis for the category of inflammatory degeneration of the dental pulp. These anaerobes have been strongly implicated in the endodontic infections that occur as sequelae to carious pulpitis. Accordingly, the data suggest that the presence of threshold levels of these bacteria in carious dentine may be indicative of irreversible pulpitis. Knowledge of the microbial predictors associated with irreversible pulpitis creates potential for the development of a diagnostic tool, and for restorative materials with antimicrobial properties.