| Summary: | Background and Objective: Tonsillar crypts can be considered a reservoir for a variety of bacterial species. Some bacterial species can be considered part of the normal oropharyngeal microbiota. The roles of other pathogens, for example, the so-called non-oral and respiratory pathogens <i>Staphylococcus aureus</i>, <i>Klebsiella</i>, <i>Pseudomonas</i>, and <i>Acinetobacter</i> spp., which have strong virulence factors, biofilm production capacity, and the ability to initiate infectious diseases, are unclear. The purpose of this study was to detect the presence of <i>S. aureus</i>, <i>K. pneumoniae</i>, <i>P. aeruginosa</i>, and <i>Acinetobacter</i> spp. within the tonsillar crypts of healthy individuals, and to analyze the pathogens’ biofilm production and antibacterial resistances. Results: Only common oropharyngeal microbiota were cultivated from 37 participant samples (40.7%). The most commonly isolated pathogenic bacterium was <i>S. aureus</i>, which was isolated in 41 (45%) participant samples. <i>K. pneumoniae</i> was isolated in seven (7.7%) samples, <i>Acinetobacter</i> spp. were isolated in five (5.5%) samples, and <i>P. aeruginosa</i> was isolated in two (2.2%) samples. Biofilm producers predominated among the pathogenic bacteria; 51 strains were biofilm producers, and among them, 31 strains were moderate or strong biofilm producers. The tested <i>S. aureus</i>, <i>K. pneumoniae</i>, <i>P. aeruginosa</i>, and <i>Acinetobacter</i> spp. strains were sensitive to commonly used antibiotics (amoxicillin–clavulanic acid, clindamycin, or ciprofloxacin). One of the isolated <i>S. aureus</i> strains was MRSA. Conclusions: Biofilm is a commonly observed feature that seems to be a naturally existing form of pathogenic bacteria colonizing human tissue. <i>S. aureus</i>, <i>K. pneumoniae</i>, <i>P. aeruginosa</i>, and <i>Acinetobacter</i> spp. occasionally occur in the tonsillar crypts of healthy individuals, and, therefore, it is most likely that <i>S. aureus, K. pneumoniae, P. aeruginosa</i>, and <i>Acinetobacter</i> spp. in opportunistic tonsillar infections originate from the tonsillar crypt microbiota.
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