Background: About 35% of the oral microbiome remains uncultured due to limitations of conventional laboratory techniques. More than 200 of those phylotypes have been catalogued in the HOMD and a subset of them had been proposed as candidate periodontal pathogens. This segment of the microbiome merits further investigation, as it might harbor important pathogens that are currently overlooked. Objective: The objective of this study was to devise an imaging approach to study such phylotypes in the conditions most conducive to their growth and begin to unveil their ecological and biogeographical characteristics. Methods: Previous work from the Teles Lab had identified the most common candidate periodontal pathogenic phylotypes and developed biofilms that fostered their growth. Such biofilms and their spent media were used for the development of the imaging approach. 16S rRNA sequencing data from 18 ex vivo biofilms developed from samples collected from 16 periodontitis patients were screened to determine the most common phylotypes. Given the unculturability of phylotypes, pure and mixed cultures of reference strains (Actinomyces israelii, Porphyromonas gingivalisand Fusobacterium nucleatum) were used to develop the method. Specific and eubacterial probes targeting 16S rRNA of the taxa of interest were synthesized and tested on pure and mixed cultures and on ex vivo biofilm samples. Fixation and permeabilization protocols were tested and optimized.Biofilm and media samples were visualized using confocal (Leica SP8 and Zeiss LSM 880) and epifluorescence (Leica DM6000B) microscopes. Results: The microbial screening of 1311 samples from 16 periodontitis patients showed that Megasphaera HOT 123, Prevotella HOT 526, Prevotella HOT 315, Aggregatibacter HOT 898and Alloprevotella HOT 912were the most prevalent and abundant phylotypes. Imaging of pure and mixed cultures of A. israelii, P. gingivalisand F. nucleatumand the use of positive and negative controls demonstrated the specificity of the probes used. Spent media samples were better visualized than biofilm samples. P. gingivalis and F. nucleatumcould be observed in several samples. Megasphaera HOT 123 could be clearly visualized as small cocci in media samples. Conclusions: The imaging method devised allowed the specific visualization of phylotype Megasphaera HOT 123 as cocci located in clusters within ex vivo biofilm and media samples. .