Microbiological studies of occlusal dental biofilms have hitherto been hampered by inaccessibility to the sampling site and demolition of the original biofilm architecture. The aim of the present study was to explore the spatial distribution of bacterial taxa in vivo at various stages of occusal caries, applying a new methodology involving preparation of embedded hard dental tissue slices for fluorescence in situ hybridization (FISH) and confocal microscopy. 11 extracted teeth were included in the study and classified according to their occlusal caries status (active/inactive/sound; cavitated/non-cavitated). The teeth were fixed (paraformaldehyde 3 %), embedded (Technovit 8100), sectioned and decalcified (EDTA 17%, pH 7.0) before FISH was performed using oligonucleotide probes for the most abundant species/genera associated with occlusal caries including Streptococcus and Actinomyces. The findings were related to histological features of lesion penetration. The sites showed distinct differences in the bacterial composition and fluorescence intensity between different ecological niches in occlusal caries. Biofilm observed along the entrance of fissures showed an inner layer of microorganisms organized in palisades often identified as Actinomyces, covered by a more loosely structured bacterial layer consisting of diverse genera, similar to supra-gingival biofilm. Biofilm within the fissure proper seemed less metabolically active, as judged by the low fluorescence signal intensity and the presence of material of non-bacterial origin resembling developmental protein, calculus and/or dead bacteria. Bacterial invasion with penetration into the dentinal tubules was seen only at advanced stages of the caries process with manifest cavity formation. It is concluded that the new methodology represents a valuable supplement to previous methods for the study of microbial ecology in caries by allowing analysis of the structural composition of the undisturbed biofilm in caries lesions in vivo.