Background: The recognition of human blood microbiota, consisting of cell wall-deficient microbes (L-forms), is a major challenge today in the field of microbiology. There are accumulating data confirming the concept of "internal" blood L-form microbiota and its significance for health and diseases. Finding out whether the blood microbiota can be of diagnostic and prognostic importance for detection and evaluation of chronic infections anywhere in the body is a major objective. In the context of chronically infected upper respiratory tract (URT), the aim of the current study was to understand whether a local infection can be a source for entry of bacteria and fungi in the blood. Methods: Blood samples from six persons with chronic inflammations in URT diagnosed with hypertrophied adenoids, chronic sinusitis, nasal polyps, chronic naso-pharyngitis and one control healthy person were studied. Blood microbiota assessment methodology that be used, included three phases: 1) isolation of L-form cultures from blood-development and propagation; 2) cultivation directed to conversion of L-forms into bacterial and fungal cultures; 3) isolation of pure classical bacterial and fungal cultures and their identification by MALDI-TOF method. Results: From the patients were isolated L-forms of opportunistic bacteria (Streptococcus mitis, Roseomonas mucosa, Dermacoccus nishinomiyaensis, Enterococcus faecalis, Acinetobacter johnsonii, Pseudomonas putida, Staphylococcus aureus, Pseudomonas luteola, Enterobacter cloacae) and fungi such as Rhodotorula mucilaginosa, Aspergillus niger, Aspergillus fumigatus and Mucorales. Conclusion: The novel innovative methodology for assessment of blood L-form microbiota was successfully applied for detection of microbes responsible for chronic infections at URT.